A PROJECT REPORT ON

Detection of sexually transmitted infectious agents using light amplification technology and pCR
SUBMITTED TO

FOR THE PARTIAL FULFILLMENT OF INDUSTRIAL INTERNSHIP (2011)

Submitted By SIMRANDEEP KAUR REGISTRATION NO: 10800802

UNDER SUPERVISION OF:

Mr. Prem Raj Singh(scientist)

WORK CARRIED OUT AT:-

AUROPROBE LABORATORIES.LTD., NEW DELHI-110024, INDIA WEBSITE www.auroprobelabs.com

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DECLARATION
I do hereby declare that the thesis work entitled detection of sexually transmited infectious agents using Light amplification technology and PCR(polymerase chain carried out at AUROPROBE

reaction) is an authentic record of my own work

LABORATORIES as required for industrial internship project for the award of degree of B.tech(hons)biotechnology ,lovely professional university(phagwara) under the guidance of Mr.prem raj singh(scientist) during july to november 2011

Simrandeep kaur Reg no:10800802

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ACKNOWLEDGEMENTS
I would also like to express my sincere and special appreciation to Dr. Nimrat Bawa, Director, Auroprobe Laboratories, Ghaziabad, Uttar Pradesh, for providing me all the laboratory facilities.

I would like to thank Mr.prem raj singh. I wish to express my sincere gratitude for his dedicated and generous support in all aspects of my work. I am grateful for his valuable scientific advice, constructive criticism, encouragement, deep commitment and guidance throughout the study.

I would like to thank and express my sincere gratitude to Mr. Narotam Sharma(scientist), Mr. nishant, Dr.parshant Scientist (training co-ordinator) Auroprobe Laboratory for his benevolent guidance and support. I extend my special and sincere thanks to all my friends for their whole hearted support, nice company and helpful suggestion.

Words fail to express my sincere regards to my parents and elders for their constant inspiration, which helped me to complete this work.

Simrandeep kaur

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LIST OF ABBREVIATIONS: % : Percentage. g : Microgram. l : Microlitre. bp : Base pairs. CSF : Cerobro Spinal Fluid. DNA : Deoxyribonucleic Acid. Ds : Double strand. e.g. : Example Gratia.(Latin: For example) EDTA : Ethylene Diamine Tetra Acetic Acid. et.al. : And others. Fig : Figure. g : Gram. HIV : Human Immunodeficiency Virus. HSV : Herpes Simplex Virus. IC : Internal Control. kb : Kilo base. l :Litre.. M : Molar. min : Minutes. ml : Millilitre. nm : Nanometer. C : Degree Centigrade. PBS : Phosphate Buffer Solution. PCR : Polymerase Chain Reaction. pH : Potential Hydrogen. rpm : Revolution per minute. TAE : Tris Acetate EDTA. TBE : Tris Borate EDTA. TM : Trade Mark. V : Voltage. MN : Macherey-Nagel. ALT : Alanine transferase B : Base(s) cDNA : Complimentary deoxyribonucleic acid CRP : C-reactive protein DNA : Deoxyribonucleic acid dNTPs : Deoxy-Nucleotide Triphosphates E.coli : Escherichia coli EDTA : Ethylene diamine tetra-acetic acid EIA : Enzyme Immunoassay ESR : Erythrocyte sedimentation rate Ex-buffer : Extension-buffer FC : Flow Cytometry IBD : Inflammatory bowel disease IU : International Units Kb : Kilobite Kd : Kilodalton MMX : Master Mix 5|Page

MQ mRNA PCR RNA RS RT SSP ss UV WHO

: Milli Q water : Messenger RNA : Polymerase Chain Reaction : Ribonucleic acid : Reiters syndrome : Reverse Transcription : Specific sequence priming : Single stranded : Ultra Violet : World Health Organization

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LIST OF FIGURES: Fig.1: Hsv Structure. Fig. 2: The Hsv Capsid. Fig. 3: Hsv Genome Structure. Fig. 4: Initial Steps In Infection- Virus Entry. Fig. 5: A Simplified Hsv Replication. Fig. 6: Viral Assembly. Fig. 7: Hsv Infection. Fig. 8: Viral Envelopment And Release. Fig. 9: Diagnosis Of Hsv Fig 10 :3d Representation Of Human Papillomavirus Fig 11: Genomic Organization Of The Hpv Genome Fig 12. Life Cycle Organization During Productive Infection By Hpv Type. Fig 13 : Virus Disable Supressor. Fig 14 : Virus Uncoats. Fig 15: Diagram Representing Penetration Of Virus In Cervix. Fig 16: 3d Structure Of Chlamydia Trachomatis. Fig 17: Showing Life Cycle Of C.Trachomatis Fig 18 : Infection By Neisseria. Fig 19: Instrument And Reagents Of Hybrid Capture Assay.

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LIST OF TABLES: 1. ZR Viral DNA Kit 2. Condition of HSV PCR amplification. 3. Reagents required for electrophoresis: 4. Shows The Characteristics Of Patients Showing Age Group Distribution Of HPV. 5. Prevalence Rate For HSV 6. Rate of occurrence of HSV in male and female among positive patients. 7. Determination of Positivity rate in age groups 8. Prevalence Rate For CT 9. Determination Of Positivity Rate In Age Groups Of CT 10. Prevalence Rate For NG 11. Determination of Positivity rate in age groups of NG 12. Analysis Of Positive Samples For HPV & HSV Viral Disease 13. Analysis Of Positive Samples For CT/NG Bacterial Disease 14. Age wise distribution of positive samples in HSV and HPV

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CONTENT 1. INTRODUCTION 2. REVIEW OF LITERATURE 2.1. HERPES SIMPLEX VIRUS 2. 2. HUMAN PAPILLOMAVIRUS 3. CT/NG 4. MATERIALS & METHODS 5. RESULTS AND DISCUSIONS 6. CONCLUSION 7. REFERENCES

PAGE NO
11-13

14-24 25-42 43-60 61-77 78-85 86-87 88-92

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OBJECTIVES

The present investigation was done to determine the prevalence of,human papillomavirus (HPV), Herpes simplex virus (HSV-1 & HSV-2),(CT/NG) in Delhi NCR region using polymerase chain reaction and light amplification technology, of different clinical samples brought in our laboratory with the following objectives. Collection of various samples from different hospitals of Delhi. Molecular Diagnosis 1. Genomic DNA extraction. 2. Amplification of DNA obtained. To study the prevalence and detection limit in different clinical samples by PCR and hybrid capture technology. To evaluate the rate of occurrence of HSV, HPV, CT/NG in different age groups.

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1. INTRODUCTION
The term sexually transmitted infections (STI) encompasses infections caused by a broad range of pathogens, including viruses, bacteria and protozoa. The transmission route of these infections is sexual contact between human beings. The number of known STI is about thirty and the prevalence of chronic viral STI such as herpes simplex virus infections and human papilloma virus infections is very high in the adult populations world-wide. The highest incidence rates of STI are generally found in urban populations between the ages of 15 and 35. It is not unlikely that the majority of adults in the world have one or more STI . Since many of these infections are asymptomatic, the term STI is preferable to the formerly used STD (sexually transmitted diseases)[1]

1.1) VIRAL AGENTS

Herpes simplex virus (HSV) is a double stranded DNA virus that belongs to Herpesviridae family. Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are two species of this family. They are also called Human Herpes Virus 1 and 2 (HHV-1 and HHV-2).The HSV genome encodes for over 80 proteins. They have particle morphology and ability to produce the disease following the infection and enter a latent phase in some cells of the infected natural host, this latent phase ensures survival of viral genome through the lifetime of the particular infected individual . It has been recognized as a relatively common human pathogen. A broad spectrum of diseases has been related to HSV-1 infection. some of these are selflimiting like herpes labialis, some are mild compromising normal function of involved organs such as keratoconjunctivitis, but others like herpes s encephalitis are life-threatening. Scientists discovered a link between HSV-1 and Alzheimers disease in 1979 [2].

Herpes simplex virus type 2 (HSV-2) infection is increasingly regarded as the most common cause of genital ulcer disease worldwide . In developing countries, the major public health importance of HSV-2 relates to its potential role in facilitating human immunodeficiency virus (HIV) transmission. HSV-2 seems to be highly prevalent in most regions with severe HIV epidemics
[3]

. Genital ulcer disease is known to enhance the infectiousness of HIV-

positive subjects and the susceptibility of HIV negative subjects, and clinical research has shown effects of herpes on genital HIV shedding. The reciprocal effect of HIV immune suppression on the exacerbation of HSV-2 symptoms implies that there may be a positive

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feedback loop, with HIV enhancing HSV-2 expression which in turn may enhance HIV transmission [4]. Accurate identification of persons with genital herpes is necessary for optimal patient management and prevention of transmission. Because of inherent inaccuracies, clinical diagnosis of genital herpes should be confirmed by laboratory testing for the causative agents herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2). Further identification of the HSV type is valuable for counselling on the natural history of infection and risk of transmission [5]. HSV-1 and HSV-2 are very closely related viruses, which have preferences for oral and genital mucosa, respectively. Independently these two virus have very similar modes of infection, varying more in their specific binding motifs but using related structures. The differences in the nucleotide sequences of their genomes and the amino acid sequences of their proteins reflect the difference in the environments in which they function. There is a need to differentiate between HSV-1 and HSV-2 infections because there are important differences between the two viruses in terms of epidemiology, natural history, clinical implications and outcome. The primary difference between the two viral types is in where they typically establish latency in the body- their "site of preference." HSV-1 usually establishes latency in the trigeminal ganglion, a collection of nerve cells near the ear. From there, it tends to recur on the lower lip or face. HSV-2 usually sets up residence in the sacral ganglion at the base of the spine. From there, it recurs in the genital area. HSV-1 is usually mild, especially when it infects the lips, face, or genitals. In some cases type 1 can recur spontaneously in the eye, causing ocular herpes, a potentially serious infection which can lead to blindness. In very rare cases HSV- 1 can spread spontaneously to the brain, causing herpes encephalitis, a dangerous infection that can lead to death. HSV-1 is also the usual cause of herpes whitlow, an infection on the finger, and "wrestler's herpes," (herpes gladiatorum) herpes infection on the chest or face. Just how much of a physical problem HSV poses for a person depends largely on three factors. The first is how well the person's immune system is able to control the infection, second how long a person has had the infection and lastly, whether the virus is established in its site of preference. Both are most contagious during active outbreaks, but are often spread through viral shedding when there are no recognizable symptoms. Genital herpes are presently incurable, although the guanine analogue acyclovir, given orally and also applied topologically, is effective in limiting the shed of active virus from blisters and promoting the healing of blistering lesions. 12 | P a g e

Following the introduction of molecular biological methods into virological diagnostics, polymerase chain reaction (PCR) has become the method of choice for the laboratory diagnosis of herpes simplex encephalitis (HSE) [5]. In the clinical laboratory setting it is of the utmost importance to optimize and make suitable diagnostic procedures according to its conditions and limitations. The powerful sensitivity of PCR makes it suitable in the diagnose of diseases in clinical laboratories, even if the amount of pathogenic agents involved is scanty and traditional procedures lack of sensitivity
[6]

. Further identification of the HSV type is

valuable for counselling on the natural history of infection and risk of transmission. Laboratory methods include antigen detection, culture, polymerase chain reaction (PCR) and conventional and type-specific serology (TSS). PCR has, by far, the greater sensitivity and should be the test of choice for symptomatic cases. Reliable methods for detection and sub-typing of HSV infections have included enzymelinked immunosorbent assay (ELISA), immunofluorescence microscopy (IFA) and virus isolation by cell culture. All three assays are laborious and time consuming, which is a limitation. The sensitivities of these techniques have also been questioned, particularly in reference to more recent methodologies, such as polymerase chain reaction (PCR). The extracted nucleic acid is used as a reactant in PCR from clinical specimens to determine if the DNA sequences of interest (i.e. an infectious agent) are present. Extractions of DNA are deemed necessary due to both assumption and empirical observation. The cross contamination of nucleic acid specimens with biological and chemical factors can inhibit PCR. Polymerase chain reactions require evaluation on a case-by-case basis to determine their efficiency. Today in countries like India, PCR machines (thermal cyclers) are more widely available than fluorescence microscopes in most diagnostic virology laboratories [7].

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2. REVIEW OF LITERATURE 2.1 Herpes simplex virus General aspects 2.1.1 Historical Background Herpes Virus Infections have been prevalent as early as ancient Greek times. Hippocrates is known to have described the cutaneous spreading of herpes simplex lesions and scholars of Greek civilization define the greek word "herpes" to mean "to creep or crawl" in reference the spreading nature of the herpetic skin lesions. Even Shakespeare is thought to have been familiar with recurrent herpes simplex lesions and their transmission. In Romeo and Juliet, he writes Queen Mab to say "O'er ladies lips, who straight on kisses dream, which oft the angry Mab with blisters plagues, because their breaths with sweetmeats tainted are." Nonetheless, it was not until 1893 when Vidal recognized that human transmission of Herpes Simplex infection from one individual to another. During the twentieth century, HSV research blossomed. Histopathologic studies characterized the multinucleated giant cells associated with herpesvirus infection. And in 1919, Lowenstein confirmed experimentally the infectious nature of HSV that Shakespeare had only suspected. In the 1920's and 1930's, the natural history of HSV was widely studied and it was found that HSV not only infects the skin, but also the central nervous system. In the 1930's, host immune responses to HSV were thoroughly examined and the property of HSV known as latency was characterized. By the 1940's and 1950's, research abounded on the many diseases caused by HSV. More recent research has focused on antiviral research, differences between HSV strains, and using HSV vectors for use in vaccines. 2.1.2 Classification Group: ds DNA. Family: Herpesviridae. Subfamily: Alphaherpesvirinae. Genus: Simplexvirus. Species: Herpes simplex virus 1 (HWJ-1), Herpes simplex virus 2 (HWJ-2).

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2.1.3 Taxonomy Herpes simplex viruses (HSV) are important human pathogens causing diseases in a variety of different tissues and animal species. There are two antigenic types, HSV-1 and HSV-2, with HSV-1 being most often transmitted non-sexually and HSV-2 most usually sexually transmitted
[8]

. HSV belongs to the family Herpesviridae. The family is divided into three

subfamilies; (alpha), (beta) and (gamma) herpesviruses, based on biological properties. At present nine herpesviruses are recognized as natural human pathogens. Herpes simplex virus types 1 and 2 (HSV-1 and HSV- 2) and Varicella-zoster virus (VZV) belong to the alphaherpesviruses (alphaherpesvirinae), which have a wide host range, a relatively short life cycle and establish latent infections, preferentially in sensory ganglia. Human cytomegalovirus (HCMV), and human herpes viruses 6A, 6B and 7 (HHV-6A, HHV-6B and HHV-7) belong to betaherpesviruses (betaherpesvirinae), which have a restricted host range and multiplication of the viruses appears to be slow. These viruses establish latency in lymph reticular cells. Finally Epstein-Barr virus (EBV) and human herpesvirus 8 (Kaposis sarcomaassociated herpes virus) are B lymphotropic virus belonging to the gammaherpesviruses (gammaherpesvirinae), and latency has been detected in lymphoid tissues [8].

2.1.4 Structure Studies have shown that all herpes virions consist of four elements: DNA core, capsid, tegument and glycoprotein-containing envelope. The HSV genome contains approximately 152-kbp.The DNA of HSV-1 and HSV-2 consist of two covalently linked segments called the L (long) and S (short), with unique sequences-UL (unique long) and US (unique short), flanked by large inverted repeat sequences which are designated terminal and internal repeats of the long (TRL and IRL) and short (TRS and IRS) unique sequences, respectively. Additionally, the unique L and S components can invert relative to one another, yielding four linear isomers, and each of the four is equally virulent (functionally equivalent) in the host cell.The genes of the long and short unique sequences are designated UL1 to UL56 and US1 to US12, respectivel.[9]

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Fig.1: HSV Structure.

Fig. 2: The HSV Capsid The capsid is a structurally well-defined icosahedron, protecting the viral genome. It is 125 nm in diameter and approximately 15 nm thick, and it is organized into 162 capsomers, of which 150 are hexavelent capsomers (hexons), and 12 pentavalent capsomers (pentons). All capsomers are connected in groups of three by trigonal nodules or triplexes on the capsid surfaces . Three types of capsids are reported . The C capsid are mature forms and contain viral DNA. The B capsids do not contain DNA, but do contain the scaffold proteins and are believed to be an early stage of viral assembly. The A capsids are empty. These are thought to be capsids that failed in the packaging process. In cell free system a fourth form of capsids has been identified, termed procapsid and this may be a precursor of these three capsids
[10]

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The tegument contains at least 19 different HSV proteins. While the precise functions of most of the individual tegument proteins are not yet clear, it seems likely that they perform dual roles in the virus replication cycle, providing activities both at the onset of infection, as the capsid-tegument enters the cell, and during virion assembly, as virus matures and exits the cell. UL 31 and UL 34 tegument proteins form a complex which plays an important role in envelopment of nucleocapsids
[11]

. UL 41 has been implicated in virally induced host-cell

shut off by degradation of host mRNAs soon after infection . The UL48 protein is the transinducing factor, TIF, responsible for the trans-activation of immediate early genes. It is also reported that UL48 plays a direct role in virion assembly and egress . The UL 46 and UL 47 proteins modulate the activity of the TIF protein . The UL6, UL15, UL17, UL25, UL28, UL32, and UL33 proteins are required for cleavage and packaging of viral DNA . On the outer side of the tegument is the envelope, which is a lipid bilayer derived from the host cell. HSV specifies at least 12 glycoproteins designated gB, gC, gD, gE, gG, gH, gI, gK, gL, gM and gJ and gN . These glycoproteins function in several important roles, including pHindependent virus entry via fusion of the virion envelope with cellular membranes , egress of infectious virion particles, cell-to-cell spread, virus induced cell fusion and immune evasion. Antigenic specificity is provided by gG, with the resulting antibody response allowing for the distinction between HSV-1 (gG-1) and HSV-2 (gG-2) [12].

2.1.5 HSV Genome Structure : The ends of the linear molecules-the "a" sequences. These are important in both circularization of the viral DNA, and in packaging the DNA in the virion. The 9,000 bp long repeat (RL), which encode both an important immediate e promoter of and most of the "gene" for the latency associated transcript (LAT). The long unique region (UL), which is 108,000 bp long, encodes at least 56 distinct proteins (actually more because some ORFs are spliced and expressed in redundant ways). It contains genes for the DNA replication enzymes and the capsid proteins, as well as many other proteins. The 6,600 bp short repeats (RS very powerful transcr
L)

to stimulate

the infected cell for all viral gene expression that leads to viral DNA replication. The origins of replication. The oriL is in the middle of the UL region. The oriS is in the RS and thus, is present in two copies. All sets of ori's operate during infection to give a very complicated replication complex--very similar to that seen in the replication of phage T4. The 13,000 bp

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unique short region (US) encode 12 ORFs, a number of which are glycoproteins important in viral host range and response to host defense.

2.1.6 Regulation of gene expression During productive infections, transcription of the viral genome occurs in a cascade-like fashion resulting in immediate-early, early, and late viral mRNAs. The (immediate-early) genes, require no prior viral protein synthesis for their expression and are responsible for the expression of the other genes in a regulated way. (early) genes, whose expression is totally independent of viral DNA, encode proteins and enzymes which are directly involved in DNA synthesis and nucleotide metabolism. The beta () genes induce the activation of the last group of genes, the gamma () or late genes, coding for many of the structural proteins in the HSV virion, including capsid proteins, which are translated in the cytoplasm and then imported into the nucleus where capsid assembly occurs [13].

Fig. 3: HSV Genome structure. HSV genome contains approximately 152-kbp DNA, it codes for 80 proteins.

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2.1.7 Entry of HSV into host cells The entry of HSV requires binding of virus to receptors on the cell surface and fusion of the virion envelope with the cell plasma membrane. The initial attachment is mediated through viral glycoprotein C (gC) and/or gB to cell surface heparan sulfate proteoglycans. The fusion of the viral envelope with the plasma membrane requires gB, gD, gH, and gL . Three cell surface receptors for HSV have been identified: herpes virus entry mediator A (HveA or HVEM) is a member of the tumor necrosis factor receptor (TNFR) family. It is also reported that HveC allows viral entry by directly interacting with gD, as shown for HveA . Recently both HveB and HveC were found to be components of a novel cell-cell adhesion system, and to belong to the Ig superfamily. They were therefore renamed as nectin-1 and nectin-2, respectively [14].

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Fig. 4: Initial Steps in Infection- Virus Entry. (a) HSV binds to the cell surface receptors, (b) followed by binding of the viral envelop with the plasma membrane and (c) the capsid attach to the nuclear envelope and the viral DNA penetrates into the nucleus.

2.1.8 Life cycle of HSV HSV replicates by three rounds of transcription that yields: immediate-early () proteins that mainly regulate viral replication; early () proteins that synthesis and package DNA; and late () proteins, most of which are virion proteins.

Fig. 5: A simplified HSV replication

2.1.9 HSV infections HSV is natural pathogens for humans, with particular affinity for the nervous tissue. The virus spread from person to person by infected secretions, classically oral secretions for HSV1 and genital secretions for HSV-2. There are three types of herpetic infections: lytic infection, latent infection and transforming infection. In a lytic infection, virus multiply inside the nucleus of infected cell. This is followed by production of infectious virions before lysis of infected cells, partly due to suppression of host protein synthesis by a structural 20 | P a g e

protein named virus host shutoff (vhs) protein, encoded by the UL41 gene (Mettenleiter, 2004). In latent infection, viral DNA is maintained in a non-replicative state and persists in the nucleus as an episome for the entire life of the individual. Virus may reactivate following a variety of local or systemic stimuli to cause recurrent disease . During latency, the viral lytic genes are extremely repressed and only a single transcription unit encoding the latencyassociated transcripts (LATs), remain active . The most abundant LAT is a 2.0-kb RNA
[15]

which is also detected during productive infections . The other LATs are 1.4- and 1.5-kb long which can only be detected during latency . Both HSV-1 and HSV-2 are able to transform the morphological phenotype of rodent cells. Failure to detect viral DNA in transformed cells led to the hit-and-run hypothesis of HSV-1 transformation. Within the HSV-2 genome there are two unique morphological transforming regions designated as mtr II and mtr III located between map units 0.585 and 0.63 and 0.42 and 0.58. MtrII and mtrIII encompass for the large subunit of viral ribonucleotide reductase (RR1) which has transforming potential. It has been proposed that HSV-2 participates as a cofactor in the development of invasive cervical carcinoma . No human tumours has so far been shown to be directly caused by HSV [16].

Fig. 6: Viral Assembly. The genes codes for proteins which assemble to form procapsid. The DNA is packaged into mature capsid.

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Fig.7: The overall HSV infection. (1) It starts by productive infection followed by latent infection which leads to the LAT gene expression (2) Immediate early transcription leads to genome replication (3), it also involves early proteins (4), viral genome replication and modification of nuclear structure occurs(5), capsid assembly takes place(6), finally the virus is enveloped and released.

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Fig. 8: Viral Envelopment and release. The viral capsid is enveloped by the nuclear envelope with the help of glycoproteins, it attaches to cell membrane and is released outside.

2.1.10 Treatment There is currently no curative therapy for HSV infection. In fact, no drugs are available acting on the virus during latency in the dorsal root ganglia. The yet most successful drugs such as acyclovir, valacyclovir, famciclovir are all nucleoside analogues inhibiting replication. These drugs have similar modes of action. They first undergoes monophosphorylation by virally encoded thymidine kinase (TK) and concentrates in infected cells. Host enzymes, cellular kinases further phosphorylate the drug to the active triphosphate form which inhibits viral DNA polymerase and consequently viral replication.Foscarnet is a pyrophosphate analogue which directly inhibits viral DNA polymerase without prior activation by thymidine kinase. In contrast to nucleoside analogues, foscarnet is a noncompetitive inhibitor of the DNA polymerase
[17]

. Various vaccine against HSV infection

have been tested without success, focusing on primary prevention and on immunotherapy for those already infected. Research is still going on for an effective vaccine [18]. 23 | P a g e

2.1.11 Diagnosis of HSV infection Efficient laboratory testing is an essential component for management and development of strategies to prevent transmission of HSV infection. Current laboratory methods used to diagnose HSV infections include: virus detection, antigen detection, DNA detection and serological tests. Virus detection methods through culturing and DNA detection, particularly using polymerase chain reaction (PCR), are applicable during active infection in patients presenting with lesion. Antigen detection method can be nearly as sensitive as culture methods
[19]

, and the most sensitive strategy is to perform both tests. Serological tests allows

identification of silent carriers of HSV infection and provide useful information in symptomatic patients when virological tests such as culture, antigen detection and PCR are not helpful. The application of HSV type specific serological tests has been difficult due to strong serological cross-reactivity caused by the extensive antigenic similarities between the two viruses. The identification of type-specific glycoproteins G-1 (gG-1) and gG-2 in the mid- 1980s seemed to resolve this difficulty , because it is antigenically distinct for the two viruses. Since the demonstration of two antigenic types, numerous test formats have been developed to detect type specific antibodies[20].

Fig. 9: Diagnosis of HSV.

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2. 2.1) HUMAN PAPILLOMAVIRUS 2.2.1) HISTORY Papillomaviruses were first identified in the early 20th century, when it was shown that skin warts, or papillomas, could be transmitted between individuals by a filterable infectious agent. In 1935 Francis Peyton Rous, who had previously demonstrated the existence of a cancer-causing sarcoma virus in chickens, went on to show that a papillomavirus could cause skin cancer in infected rabbit.

A German scientist has won the Nobel Prize in Medicine, for his discoveries about the family of viruses that cause cervical cancer.

Harald zur Hausen, MD, bucked conventional medical thinking in the 1970s and pursued the idea that HPV, or human papilloma virus, played a role in cervical cancer. Eventually, he singled out HPV 16 and 18, the strains responsible for about 70% of cervical cancers worldwide.

In

1972,

the

association

of

the

human

papilloma

viruses

with skin

cancer in epidermodysplasia verruciformis was proposed by Stefania Jablonska in Poland. In 1978 Jablonska and Gerard Orth at the Pasteur discovered HPV-5 in skin cancer. In 1976 Harald zur Hausen published the hypothesis that human papilloma virus plays an important role in the cause of cervical cancer. In 1983 and 1984 zur Hausen and his collaborators identified HPV16 and HPV18 in cervical cancer.

2.2.2) cervical carcinoma and HPV: Human Papillomavirus: A Virus that Affects Both Men and Women. The human papilloma viruses are small DNA tumor viruses that belong to the family of Papovaviridae. The incidence of cervical carcinoma worldwide is estimated to be as high as 400,000 diagnoses per year. More than 90 percent of high-grade cervical dysplasias and invasive cervical cancers have been associated with 10 to 15 high-risk HPV viral types.

Most HPV viral types are associated with sexually-transmitted infections that usually resolve spontaneously. The HPV DNA Test uses a combination of DNA probes to detect high-risk 25 | P a g e

viral types that are potentially carcinogenic (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68).

Genital HPV infection is thought to be the most common sexually-transmitted disease; males are carriers of HPV on the penile skin. Interestingly, penile carcinomas appear to be related to HPV in only a small fraction of cases, in contrast to carcinomas of the cervix. Research has shown that HPV infections of the cervix may follow a number of different pathways . Human papillomavirus is a family of Papillomaviridae.It is double stranded DNA.[21]

FIG 10 :3D Representation Of Human papillomavirus 2.2.3) Human papillomavirus infection: The most important risk factor in the development of cervical cancer is infection with a highrisk strain of human papillomavirus. Even though HPV is an important risk factor for cervical cancer, most women with this infection do not get cervical cancer and doctors believe other risk factors must come into play for this cancer to develop. Having unprotected sex, especially at a young age, makes HPV infection more likely. Women who have many sexual partners (or who have sex with men who have had many partners) have a greater chance of getting HPV.

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More than 60 types of HPV are acknowledged to exist (some sources indicate more than 200 subtypes). Of these, 15 are classified as high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), 3 as probable high-risk (26, 53, and 66), and 12 as low-risk (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, and CP6108), but even those may cause cancer. Types 16 and 18 are generally acknowledged to cause about 70% of the cancer cases.

Although most HPV infections clear up on their own, the infections could increase to major abnormalities or cervical cancer. The presence of strains 16, 18 and 31 is the prime risk factor for cervical cancer, and Walboomers et al. (1999) reported that the presence of HPV is a necessary condition for the development of cervical cancer. A virus cancer link with HPV has been found to trigger alterations in the cells of the cervix, leading to the development of cervical intraepithelial neoplasia and cancer.[22]

Disease Common warts Plantar warts Flat warts Anogenital warts

HPV type 2, 7 1, 2, 4, 63 3, 10 6, 11, 42, 44 and others Highest risk: 16, 18, 31, 45 Other high-risk: 33, 35, 39, 51, 52, 56, 58, 59 Probably high-risk: 26, 53, 66, 68, 73, 82 more than 15 types 13, 32 6, 7, 11, 16, 32 16

Genital cancers

Epidermodysplasia verruciformis Focal epithelial hyperplasia (oral) Oral papillomas Oropharyngeal cancer

TABLE 2.1: Various Types Of Warts & Cancer Caused By HPV

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HPV subtypes 16 and 18 introduce the genes E6 and E7 which code for proteins that inhibit p53 and Retinoblastoma protein (Rb), which are two important tumor suppressor genes in humans. The p53 gene product is involved in regulation of apoptosis (cell suicide), and Rb is responsible for halting the cell cycle at the G1-phase. When Rb function is impaired, the cell is allowed to progress to S-phase and complete mitosis, resulting in proliferation and hence neoplastic transformation .

Genital warts are caused by different HPV types, and are not related to cervical cancer. The medically accepted paradigm, officially endorsed by the American Cancer Society and other organizations, is that a patient must have been infected with HPV to develop cervical cancer, and is hence viewed as a sexually transmitted disease, but not all women infected with HPV develop cervical cancer. Use of condoms does not always prevent transmission. Likewise, HPV can be transmitted by skin-to-skin-contact with infected areas. In males, HPV is thought to grow preferentially in the epithelium of the glans penis, and cleaning of this area may be preventative.

Despite the development of an HPV vaccine, some researchers argue that routine neonatal circumcision is an acceptable way to lower the risk of cervical cancer in men. Others maintain that the benefits do not outweigh the risks and/or consider the removal of healthy genital tissue from infants to be unethical as it cannot be reasonably assumed that a male would choose to be circumcised. There has not been any definitive evidence to support the claim that male circumcision prevents cervical cancer, although some researchers say there is compelling epidemiological evidence that men who have been circumcised are less likely to be infected with HPV. However, in men with low-risk sexual behaviour and monogamous female partners, circumcision makes no difference to the risk of cervical cancer.[23]

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FIG 11: Genomic organization of the HPV genome[24]

2.2.4) Infection Is Sexually Transmitted: Anyone who has ever had genital contact with another person infected with HPV can get the infection and can pass it to another person. Since the virus can be silent for many years, a person can have genital HPV even if years have passed since he or she had sex. 2.2.5) Diversity amongst human papillomaviruses : Papillomaviruses are a diverse group of viruses that have been found in more than 20 different mammalian species, as well as in birds and reptiles. because of their medical importance, the human papillomaviruses (hpv) have been most extensively studied, and more than 100 different types have now been identified .although papillomavirusclassification is based on nucleotide sequence homology, the differences between evolutionary groups are reflected to some extent, in the differences that exist in the biology of the different viruses. genitally transmitted hpv types are contained within supergroup a (also known as alpha papillomaviruses) and viruses from this group, such as hpv6 and 11, are major sexually transmitted pathogens that arethought to affect around 1% of the sexually active population 29 | P a g e

.these viruses can also infect oral sites where they are generally associated with benign papillomas.by contrast, the high-risk viruses from supergroup a, such as hpv16 and 18, cause mucosal lesions that can progress in some individuals to high-grade neoplasia and cancer .although viruses from supergroup a also include members whose primary tropism is for cutaneous sites, such as hpv2 or hpv10, these viruses share life cycle features that do not extend to papillomaviruses from other evolutionary groups. hpv2 and closely related supergroup a papillomaviruses are the primary cause of common warts. The second major group of human papillomaviruses are contained within supergroup b. viruses from the b1 subgroup such as hpv5 (also known as beta papillomaviruses) cause inapparent or latent infections in the general population, but can become a problem in immuno-suppressed individuals and in individuals who have an inherited defect which renders them susceptible to infection by papillomaviruses from the b1 supergroup. such patients can develop skin cancers at the site of hpv infection, and it is thought that b1 viruses may be involved in the development of non-melanoma skin cancer (nmsc) in the general population. by contrast, viruses from the b2 subgroup such as hpv4, cause cutaneous warts in the general population that can superficially resemble those caused by supergroup a papillomaviruses such as hpv2. the remaining group of hpvs are contained within supergroup (also classified as mu and nu-papillomaviruses .Only three human members from this group are known, and all cause cutaneous papillomas in the general population. hpv1 is the most well studied member of this group, and like hpv2 in supergroup a, causes verrucas and palmar warts.[25]

2.2.6) Problems in developing a general model of hpv-associated disease It is apparent from the above overview that different hpvs have evolved to fill different biological niches, and that in some instances, viruses from different evolutionary groups may be able to target the same epithelial site. Despite this apparent heterogeneity amongst hpvs, they all share certain features that allow them to produce infectious virions following infection. all known hpvs are exclusively epitheliotropic, and unlike certain animal papillomavirus types such as bovine papillomavirus type 1 (bpv1) or bpv2, they do not infect or express their gene products in the underlying dermis. similarly, all produce infectious particles in the upper epithelial layers, although there appear to be differences in the extent of virus synthesis depending on whether transmission is through direct contact (e.g., genital warts), or whether it occurs indirectly (e.g., verrucas). while we have a basic understanding of how papillomaviruses cause disease, it is becoming apparent that the evolutionary 30 | P a g e

backgrounds of the different viruses, their site of infection, and their mode of transmission must all be considered if the general model is to be applied to particular hpv types. Differences in regulatory sequences and coding potential within the viral genome are likely to underlie the significant differences that are apparent in the biology of different papillomavirus types.

2.2.7) Organization Of The Hpv Life Cycle Most work on hpvs has centred on the analysis of thehigh-risk hpv types and in particular on hpv16, which is the primary cause of cervical cancer from these studies and from the analysis of related hpv types (including hpv11 and hpv1), a general pattern of viral gene expression has been worked out that can, with modification, be applied to human papillomaviruses from other groups.

2.2.8) Infection and uncoating Initial infection requires access of infectious particles to cells in the basal layer, which for some hpv types is thought to require a break in the stratified epithelium. Such breaks may not be readily apparent, and may occur under conditions where the skin is exposed to water or is abraded (e.g., swimming pool surfaces), or is subjected to other environments where micro traumas may develop. it has been suggested that for a lesion to be maintained, the virus must infect an epithelial stem cell. in cutaneous skin, such stem cells are abundant within the hair follicle, and for viruses of the b1 supergroup (which are prevalent but which cause inapparent infections), the hair follicles may represent an important site of entry. several studies have shown that dna of viruses from the b1 supergroup can be readily amplified by pcr from plucked

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FIG 12. life cycle organization during productive infection by hpv types from supergroup a. (a) diagrammatic representation of the skin to reveal the pattern of hpv16 gene expression as the infected cell migrates towards the epithelial surface. other supergroup a viruses, such as hpv2 and hpv11 follow a similar pattern. after infection (in this case through a cut), the viral genome is maintained as a low copy number episome. during epithelial differentiation, the p97 promoter directs expression of the e6 and e7 genes necessary for s-phase entry (red). The p670 promoter is up-regulated in the higher epithelial layers, and viral replication proteins (e1, e2, e4, e5) increase in abundance (green), facilitating amplification of viral genomes (blue). changes in mrna splicing allow e4 to persist into the upper epithelial layers where the viral capsid proteins (yellow) are found. (b) cells in the lower epithelial layers are s-phase competent. viral genome amplification begins in these cells but ceases once the cells lose their ability to express s-phase proteins. although amplified viral genomes can be detected throughout the upper epithelial layers, cells that are actively supporting genome amplification appear confined to a region where e7 expression coincides with the high-level expression of e4, and probably also, an increase in the abundance of e1 and e2. hair follicles. for high-risk mucosal viruses such as hpv16, the formation of cervical lesions may be facilitated by infection of columnar cells, which can subsequently go on to form the basal layer of the stratified epithelium of the transformation zone. controversy exists as to the nature of the cell surface receptor that allows initial attachment of the virus to the cell, although most studies have suggested a dependence on the presence of heparin sulphate. recent work has suggested that the internalization of bound virions is a slow process with a half-life of hours rather than minutes, and that it occurs through the endocytosis of clathrin coated vesicles. papillomavirus uncoating may be facilitated by the disruption of intracapsomeric disulphide bonds in the reducing environment of the cell allowing viral dna to be transported into the nucleus.

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2.2.9) Genome maintenance Following infection and uncoating, it is thought that the virus maintains its genome as a low copy number episome in the basal cells of the epithelium. the pattern of viral gene expression in these cells is not well defined, but it is generally thought that the viral e1 and e2 proteins are expressed in order to maintain the viral dna as an episome and to facilitate the correct segregation of genomes during cell division .failure to express the full length e1 protein in the context of the hpv31 genome, prevents episomal maintenance, and in cultured epithelial cells leads to the integration of viral genomes into the host cell chromosome .whether the viral transforming proteins (e6 and e7) are also expressed in cells of the basal layer is not certain, although it appears that initial infection is followed by a proliferative-phase that results in an increase in the number of basal cells harbouring viral episomes. the number of viral genomes, and the pattern of viral gene expression in cell lines derived from low-grade cervical lesions appears to reflect those found in the basal layer of naturally-occurring lesions. it has been suggested that the viral genome is maintained in the basal layer at around 10200 copies per cell, and that viral early proteins (e6, e7, e1 and e2) are expressed at low level. The contribution of e6 and e7 to basal cell proliferation during in vivo infection is currently uncertain, and it has been suggested that expression of e1 (and possibly also e2) may be sufficient for the basal maintenance of viral episomes.

2.2.10) Proliferative-phase In uninfected epithelium, basal cells exit the cell cycle soon after migrating into the suprabasal cell layers and undergo a process of terminal differentiation. changes include the physical cross-linking of keratin intermediate filaments, the formation of cornified envelopes, and the secretion of lipids, which together allow the epithelial surface to form a physical barrier against the environment. during papillomavirus infection, e7 (and presumably alsoe6) is expressed in these cells, the restraint on cell cycle progression is abolished and normal terminal differentiation is retarded. e6 and e7 are thought to work together to achieve these effects, and in lesions caused by high-risk hpv types (such as hpv16), the two proteins are expressed from a bicistronic mrna expressed from the viral early promoter (p97). both e6 and e7 have functions that stimulate cell cycle progression and both can associate with regulators of the cell cycle. the association of e7 with members of the pocket protein family such as prb is well characterized. Prb is a negative regulator of the cell cycle that normally prevents sphase entry by associating with the e2f family of transcription factors. e7 binding to prb displaces e2f, irrespective of the presence of external growth factors, and leads 33 | P a g e

to the expression of proteins necessary for dna replication. e7 can also associate with other proteins involved in cell proliferation, including histone deacetylases, components of the ap-1 transcription complex, and the cyclin-dependent kinase inhibitors p21 and p27. despite the ability of e7 to stimulate cell proliferation, during productive infection only a subset of cells in the parabasal layers are mitotically active. the expression of cyclin e is absolutely necessary for s-phase entry, and is expressed during natural infection as a result of e7 expression and disruption of the e2f/prb complex. in differentiating epithelial cells, however, the high levels of cyclin-dependent kinase inhibitors (p21cip1 and p27kip1) can lead to the formation of inactive complexes that contain e7, cycline/cdk2 and either p21 or p27. it appears that during natural infection, the ability of e7 to stimulate s-phase progression is limited to the subset of differentiated cells with low levels of p21/p27, or which express high enough levels of e7 to overcome the block to s-phase entry. the viral e6 protein complements the role of e7, and is thought to prevent the induction of apoptosis in response to unscheduled s-phase entry mediated by e7. although the association of e6 with p53, and the inactivation of p53-mediated growth suppression and/or apoptosis has been well documented, e6 can also associate with other pro-apoptotic proteins including and bax . as a consequence, the presence of e6 is considered a predisposing factor in the development of hpv-associated cancers, allowing the accumulation of chance errors in host cell dna to go unchecked. the e6 protein of high-risk hpv types can also stimulate cell proliferation independently of e7 through its cterminal pdz-ligand domain e6-pdz binding is sufficient to mediate suprabasal cell proliferation and may contribute to the development of metastatic tumours by disrupting normal cell adhesion. In addition to e6 and e7, it is thought that the other viral early proteins (i.e., e1, e2, e4 and e5) are expressed prior to the onset of genome amplification in order to ensure maintenance of the viral episome at low copy-number .

2.2.11) Genome amplification

For the production of infectious virions, papillomaviruses must amplify their viral genomes and package them into infectious particles. for supergroup a, hpv types such as hpv16, hpv11 or hpv2, this occurs in the mid or upper epithelial layers following an increase in activity of the late (differentiation dependent) promoter. the late promoter resides within the e7 open reading frame and it is thought that its up-regulation leads to increased expression of proteins involved in viral dna replication (i.e., e1, e2, e4 and e5) without directly affecting expression of the e6 and e7 proteins that are necessary for s-phase entry). amplification of viral genomes 34 | P a g e

begins in a subset of cells in the proliferative compartment and requires expression of all viral early gene products including e4 and e5, whose roles in replication are not yet fully understood. the binding of e2 to the hpv upstream regulatory region is necessary for viral dna replication, and recruits the e1 dna helicase to the viral origin of replication. the assembly of the e1/e2 initiation complex on the viral origin is analogous to the formation of the complex between cellular initiation proteins (cdc6 and mcms) on cellular origins, and may allow the replication of viral genomes to proceed in the absence of cellular dna synthesis. throughout the virus life cycle, the relative levels of different viral proteins are controlled by promoter usage and by differential splice site selection, with an increase in the level of e1 and e2 allowing an increase in viral copy number in the upper epithelial layers. the molecular mechanisms that lead to activation of the late promoter and up-regulation of e1/e2 expression are not yet well understood, and it remains possible that this promoter is constitutively active at all stages during the productive cycle. current models suggest that a modest increase in promoter activation during differentiation may lead to an increase in the level of e1 and e2 (and also e4 and e5), and a subsequent increase in genome copy number. the newly replicated genomes would serve as templates for the further expression of e1 and e2, which would facilitate additional amplification of viral genomes and in turn, further expression of the e1 and e2 replication proteins .

2.2.12) Virus synthesis Papillomaviruses encode two structural proteins that are expressed in the upper layers of infected tissue once viral genome amplification has been completed .l2 is a minor coat protein that like l1 is produced in a subset of the cells that express e4. the major capsid protein 11 is expressed after l2 allowing the assembly of infectious particles in the upper layers of the epithelium .Papillomavirus particles comprise an approximately 8000 base pair genome within a capsid that contains 360 copies of the l1 protein, and probably 12 copies of l2, organized into a 72 capsomere icosohedral shel. the l2 protein accumulates at nuclear structures known as pml bodies during virus assembly (possibly through association with the transcription factor daxx and recruits l1 to these domains. it has been suggested that pml bodies may be the sites of papillomavirus dna replication and that capsid proteins accumulate at these sites to facilitate packaging. although virus like particles can assemble in the absence of l2, the l2 protein is thought to enhance packaging and infectivity. to be successful, the virus must eventually escape from the infected skin cell and survive extra-cellularly prior to re-infection. papillomaviruses are non-lytic, and are not released until the infected cells reach 35 | P a g e

the epithelial surface. Papillomaviruses are resistant to desiccation and their extra-cellular survival may be enhanced if they are shed from the epithelial surface within a cornified squame .the intracellular retention of papillomavirus antigens until the cell reaches the uppermost epithelial layers may compromise the immune detection of the virus, particularly as the virus also has molecular mechanisms that limit the presentation of viral epitopes to the immune system in the lower epithelial layers. although the expression of viral proteins can inhibit expression of differentiationmarkers preventing the formation of normal cornified squames , it has also been suggested that the viral e4 protein may contribute directly to virus egress in the upper epithelial layer by disturbing keratin integrity and by affecting the assembly of the cornified envelope.

2.2.13) life cycle organization amongst hpvs of different type Although all papillomaviruses must follow the broad pattern of events described above in order to produce infectious virions, different strategies of productive infection are apparent between the different evolutionary groups. Human papillomaviruses from the b2 supergroup such as hpv4 for instance, do not contain the lxcxe motif necessary for prb association in their e7 protein, suggesting that at a molecular level they may operate differently from viruses of supergroup a, such as hpv2, which cause lesions at similar sites. similarly, the e4 protein of hpv4 appears to lack the classical keratin binding motif that is present in hpv1 (e supergroup) and hpv2 (a supergroup), despite infecting cutaneous epithelium, and sharing the same requirements as other hpv types regarding escape from the cornified squame. comparative analysis of papillomaviruses of different types has shown the e1 and l1 regions to be the most highly conserved. It appears that these orfs are fundamental for the survival of all papillomaviruses, and that they were probably present in the ancestor of modern papillomaviruses. despite the diversity amongst papillomaviruses, it appears that viruses from related evolutionary groups share certain similarities. This can be illustrated by comparing papillomaviruses contained within the e supergroup (such as hpv 1) with those from the a group (such as hpv2), both of which cause verrucas. in the former group, which includes canine oral papillomavirus (copv), genome amplification begins as soon as cells leave the basal layer, without the intervening proliferative-phase characteristic of viruses such as hpv2 or hpv11. it has been speculated that these differences may reflect differences in transmissionroutes of the different hpv types and the need to produce the appropriate number of virus particles to allow infection without stimulating immunity against

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infection. it is equally possible that the co-evolution of papillomaviruses with their hosts has led to adoption of different general strategies to achieve the same goal.[26]

2.2.14) Latency period: Once an HPV viron invades a cell, an active infection occurs, and the virus can be transmitted. Several months to years may elapse before squamous intraepithelial lesions (SIL) develop and can be clinically detected. The time from active infection to clinically detectable disease makes it difficult for someone who has become infected to establish which partner was the source of infection. 2.2.15) Virus Disables Suppressors: Viral proteins E6 and E7 then disable the normal activities of the woman's own suppressor genes, which make suppressor proteins that do "damage surveillance" in normal cells. These proteins usually stop cell growth when a serious level of unrepaired genetic damage exists. Even after suppressors are disabled in a woman's cervical cells, it usually takes more than 10 years before the affected tissue becomes cancerous .

FIG 13 : Virus Disable Supressor 2.2.16) Virus Uncoats:

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The HPV sits inside the epithelial cells housed in a protective shell made of a viral protein called L1. After the virus enters the cell, the viral coat is degraded, leading to the release of the virus' genetic material into the cell and its nucleus. From the nucleus, the genes of the virus are expressed, including two genes called E6 and E7, which instruct the cell to build viral proteins called E6 and E7.

FIG 14 : Virus Uncoats 2.2.17) Cervix: The cervix is the name for the lowest part of the uterus. The uterus is an organ that only women have, and it is where a baby grows and develops when a woman is pregnant. During pregnancy, the uterus has an enormous increase in size. When a woman is not pregnant, the uterus is a small, pear-shaped organ that sits between a woman's rectum and her bladder. The cervix connects the uterus with the birth canal (the vagina). The cervix can both be visualized and sampled by your doctor during a routine pelvic examination in his or her office.

2.2.18) Virus Penetrates Cervix:

Both harmless and cancer-linked human papillomaviruses pass by skin-to-skin contact. The high-risk types of HPVs need to penetrate deeply into the lining of the cervix to establish a chronic infection. A vaginal sore or sex, which can abrade the lining, may provide a point of entry for the papillomavirus.

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Once inside the cervical lining, the virus attaches to epithelial cells. As these cells take in nutrients and other molecules that are normally present in their environment, they also take in the virus. Over 99 percent of cervical cancer cases are linked to long-term infections with high-risk human papillomaviruses.[27]

FIG 15: Diagram Representing Penetration Of Virus In Cervix

2.2.19) DIAGNOSIS: Cervical Testi In March 2003, the U.S. Food and Drug Administration (FDA) approved a test manufactured by Qiagen, which is a "hybrid-capture" test , as the primary screening tool for detecting HPV cervical infection as an adjunct to Pap testing, and may be performed during a routine Pap smear. It can detect the DNA of the 18 HPV types that most commonly affect the cervix and distinguish between "low" and "high-risk" HPV types, but it cannot determine the specific HPV types.

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According to the National Cancer Institute, "testing samples of cervical cells is an effective way to identify high-risk types of HPV that may be present. The FDA has approved an HPV test as a follow-up for women who have an ambiguous Pap test (a screening test to detect cervical cell changes) and, for women over the age of 30, for general cervical cancer screening. This HPV test can identify at least 13 of the high-risk types of HPV associated with the development of cervical cancer. The test can detect high-risk types of HPV even before there are any conclusive visible changes to the cervical cells."

The recent outcomes in the identification of molecular pathways involved in cervical cancer provide helpful information about novel bio- or oncogenic markers that allow monitoring of these essential molecular events in cytological smears, histological or cytological specimens. These bio- or onco- markers are likely to improve the detection of lesions that have a high risk of progression in both primary screening and triage settings. E6 and E7 mRNA detection PreTect HPV-Proofer, (HPV OncoTect) or p16 cell-cycle protein levels are examples of these new molecular markers. According to published results these markers, which are highly sensitive and specific, allow to identify cells going through malignant transformation.

2.2.20) Pathologic types:

Cervical intraepithelial neoplasia, the precursor to cervical cancer, is often diagnosed on examiniation of cervical biopsies by a pathologist. Histologic subtypes of invasive cervical carcinoma include the following . squamous cell carcinoma (about 80-85%) adenocarcinoma adenosquamous carcinoma small cell carcinoma neuroendocrine carcinoma

2.2.21) Treatment And Prevention:

Microinvasive cancer (stage IA) is usually treated by hysterectomy (removal of the whole uterus including part of the vagina). For stage IA2, the lymph nodes are removed as well. An alternative for patients who desire to remain fertile is a local surgical procedure such as a loop electrical excision procedure (LEEP) or cone biopsy.

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Early stages (IB1 and IIA less than 4 cm) can be treated with radical hysterectomy with removal of the lymph nodes or radiation therapy. Radiation therapy is given as external beam radiotherapy to the pelvis and brachytherapy (internal radiation). Patients treated with surgery who have high risk features found on pathologic examination, are given radiation therapy with or without chemotherapy in order to reduce the risk of relapse.

Larger early stage tumors (IB2 and IIA more than 4 cm) may be treated with radiation therapy and cisplatin-based chemotherapy, hysterectomy (which then usually requires adjuvant radiation therapy), or cisplatin chemotherapy followed by hysterectomy.

Advanced stage tumors (IIB-IVA) are treated with radiation therapy and cisplatin-based chemotherapy. Awareness : According to the US National Cancer Institute's 2005 Health Information National Trends survey, only 40% of American women surveyed had heard of human papillomavirus (HPV) infection and only 20% had heard of its link to cervical cancer. In 2006 an estimated 10,000 women in the US will be diagnosed with this type of cancer and nearly 4,000 will die from it. Screening : The widespread introduction of the Papanicolaou test, or pap smear for cervical cancer screening has been credited with dramatically reducing the incidence and mortality of cervical cancer in developed countries. The pap smear suggests the presence of cervical intraepithelial neoplasia (premalignant changes in the cervix) before a cancer has developed, allowing for further workup. Recommendations for how often a Pap smear should be done vary from once a year to once every five years. The American Cancer Society recommends that cervical cancer screening should begin approximately three years after the onset of vaginal intercourse and/or no later than twenty-one years of age. If premalignant disease or cervical cancer is detected early, it can be treated relatively noninvasively, and without impairing fertility.

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2.2.22) Vaccination: There are two HPV vaccines: Gardasil is now available to all teens and young women 9-26 years of age. The vaccine works to prevent two types of HPV: 16 and 18, which have been linked to cervical cancer Cervarix protects against HPV types 16 and 18 and is also three shots. It is for girls and women 10-25 years old.

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3. Bacterial agents:-Chlamydia trachomatis & Neisseria gonorrhoeae

3.1) INTRODUCTION TO CT/NG Neisseria gonorrhoeae and Chlamydia trachomatis are recognized as two of the most prevalent sexually transmitted bacterial infections . Worldwide, there is an estimated annual incidence of 25 million cases of gonorrhea and 50 million cases of chlamydia. In an effort to prevent the spread of these diseases, increased attention is being focused on early diagnosis and treatment of symptomatic or asymptomatic infected individuals. In men, C. trachomatis causes 40 to 50% of cases of nongonococcal urethritis, making it one of the most common sexually transmitted diseases (STD) in heterosexual males. In women, chlamydia is a major cause of pelvic inflammatory disease leading to infertility and ectopic pregnancy and can result in conjunctivitis and pneumonia in newborn infants exposed during passage through an infected birth canal.Symptoms and complications of gonorrhea are similar to those of chlamydia, and a substantial proportion of infected individuals, especially women, are asymptomatic. For both organisms, asymptomatic persons serve as a reservoir of infection, and since coinfection is common, symptoms may overlap, making clinical diagnosis difficult. Conventional diagnosis of N. gonorrhoeae infection requires isolation on selection media or observation of gram-negative diplococci in Gram smears of genital discharge, or urethral or cervical swabs. Although culture is relatively inexpensive and highly sensitive, it is logistically complicated. A pelvic examination is required for women, and insertion of a urethral swab is required for men. Diagnosis of C. trachomatis infection is frequently based on isolation in tissue culture. This procedure requires careful specimen collection and stringent transport conditions and requires at least 48 to 72 h to perform. Similar to the routine diagnosis of gonorrhea, detection of Chlamydia usually involves an invasive procedure. Since 30 to 70% of all chlamydial and gonococcal infections may be asymptomatic, routine, noninvasive screening of individuals at risk for chlamydial or gonococcal infection is highly desirable. Treatment is relatively simple and straightforward, with a 7-day course of doxycycline twice a day or a single dose of azithromycin for C. trachomatis, and a single dose of a quinolone or cephalosporin for N. gonorrhoeae. A rapid, easily performed, and accurate diagnostic test that can be performed on noninvasive samples would facilitate early recognition and treatment of these infections and thus ultimately reduce partner infections. Recent studies have demonstrated that molecular amplification assays such 43 | P a g e

as PCR and ligase chain reaction (LCR) have high sensitivity and specificity for detection of either gonorrhea or chlamydia in a variety of sample types including urethral and endocervical swabs and urine. However, they require separate processing and amplification techniques for each pathogen. In order to improve the efficiency of specimen processing and amplification, we evaluated a duplex PCR for N. gonorrhoeae and C. trachomatis that simultaneously detects both pathogens in a single amplified swab or urine specimen.

3.1.1) CHLAMYDIA TRACHOMATIS: 3.1.1) Classification Of Chlamydia trachomatis: Class:Chlamydiae Order:Chlamydiales Family:Chlamydiaceae Genus: Chlamydia 3.1.2) History: Globally, cervical cancer is among the 3 most prevalent malignancies in women. Oncogenic types of human papilloma (wart) virus (HPV) are major risk factors for cervical cancer but by no means totally explain the epidemiology of the condition. However HPV infection is common and few such cases progress to cervical cancer, suggesting that other cofactors are involved. Identifying such co-factors is difficult, as sexually transmitted infections including HPV and C. trachomatis are subject to numerous behavioural and clinical confounder. Furthermore these may operate at different points in the long progression from initial HPV infection to cancer . An early attempt by Schachter et al., 1982 to demonstrate an association between chlamydia and cervical cancer in the era before the role of HPV was discovered did not control for sexual behaviour and, in hindsight, was inconclusive [Zenilman, 2001]. In the post HPV era, Koutsky et al., 1992 found that cervical intraepithelial neoplasia (CIN), after adjustment for HPV status, was independently associated with serological evidence of chlamydial, gonococcal and cytomegalovirus (CMV) infection.[28] 3.1.3) Chlamydia trachomatis : The Chlamydiales are bacteria that, like the Rickettsiales, are obligate intracellular parasites of eukaryotic cells. 44 | P a g e

. FIG 16: 3D Structure Of Chlamydia trachomatis Unlike the Rickettsiae, the Chlamydiales have a distinctive developmental cycle for their replication. Members of the Chlamydiales share greater than 80% sequence identity for the gene encoding their 16S ribosomal rRNA (ribosomal ribonucleic acid) and/or greater than 80% identity for the gene encoding their 23S rRNA [Everett et al., 1999]. They are found within the cells of vertebrates and amoebae, while similar particles have been reported in invertebrate species including coelenterates, arthropods and molluscs. Members of the Chlamydiales are trivially referred to as chlamydiae. Many chlamydiae coexist in an apparently asymptomatic state within hosts which probably act as a natural reservoir for them. In 1907, Halberstaedter and von Prowazek, working in Java, described the transmission of trachoma from man to orang-utans by inoculating their eyes with conjunctival scrapings. C. trachomatis is a sexually transmitted microorganism responsible for a wide spectrum of diseases that include cervicitis, salpingitis, endometritis, urethritis, epididymitis,

conjunctivitis, and neonatal pneumonia. In contrast to gonorrhea infection, most men and women who are infected are asymptomatic, and, therefore, diagnosis is delayed until a positive screening result or upon discovering a symptomatic partner. In July 2007, The US Preventive Services Task Force Screening released a new recommendation statement for chlamydial infections. 3.1.4) TRANSMISSION AND SYMPTOMS OF INFECTION: Chlamydia can be transmitted during vaginal, anal, or oral sex, and can be passed from an infected mother to her baby during vaginal childbirth. Between half and three-quarters of all women who have a chlamydia infection of the neck of the womb (cervicitis) have no symptoms and do not know that they are infected. In men, infection of the urethra (urethritis) is usually symptomatic, causing a white discharge from the penis with or without pain on urinating (dysuria). Occasionally, the condition spreads to the upper genital tract in women 45 | P a g e

(causing pelvic inflammatory disease) or to the epididymis in men (causing epididymitis). If untreated, chlamydial infections can cause serious reproductive and other health problems with both short-term and long-term consequences.[29]

3.1.5) INFECTION: The patho physiologic mechanisms of Chlamydia are poorly understood at best. The initial response to infected epithelial cells is a neutrophilic infiltration followed by lymphocytes, macrophages, plasma cells, and eosinophilic invasion. The release of cytokines and interferons by the infected epithelial cell initializes this inflammatory cascade.

Infection with chlamydial organisms invokes a humoral cell response, resulting in secretory immunoglobulin A (IgA) and circulatory immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies and a cellular immune response. Recent studies have implicated a 40-kd major outer membrane protein (MOMP), as well as 10- and 60-kd chlamydial heat-shock proteins (cHSP), in the immunopathologic response, but further studies are needed to better understand these cell-mediated immune responses.

Chlamydiae have a unique biphasic life cycle that is adaptable to both intracellular and extracellular environments. In the extracellular milieu, the so-called elementary body (EB) is found. EBs are metabolically inactive infectious particles; functionally, they are spore-type structures. Once inside a susceptible host cell, the EB prevents phagosome-lysozyme fusion and then undergoes reorganization to form a reticulate body (RB).

The RB synthesizes its own DNA, RNA, and proteins but requires energy in the form of adenosine triphosphate (ATP) from the host cell. After a sufficient amount of RBs have formed, some transform back into EBs, exiting the cell to infect others .[30]

3.1.6) LIFE CYCLE: Chlamydia trachomatis exhibits an affinity for the epithelial cells of mucosal membranes such as those found on the surfaces of the cervix, urethra, rectum, nasopharynx and conjunctiva, and enter these cells by a phagocytic process4. Within infected cells, Chlamydiae occur in intracytoplasmic vesicles, or inclusion bodies. Within these inclusion bodies, morphological development takes place and two distinct particles are 46 | P a g e

observed: a small, dense infective particle, the elementary body which is transformed in the host cell into the larger less dense form, the reticulate body. These non-infective but metabolically active reticulate bodies synthesise proteins and their own DNA and RNA, then replicate by binary fission to form microcolonies within the inclusion bodies. Between 18-24 hours post infection, the reticulate bodies divide and then ultimately some of the reticulate bodies reorganise into large numbers of elementary bodies. Between 48 and 72 hours post infection, the host cells ruptures releasing elementary bodies which can infect new host cells5. See below.[31]

FIG 17: Showing life cycle of C.trachomatis

3.1.7) DIAGNOSIS OF C.trachomatis: CULTURE : At present, culture is the preferred laboratory test. However, if the specimens require long transportation times or have been exposed to extreme temperatures, culture is less sensitive than the nucleic acid methods. Culturing isolates is important for antimicrobial susceptibility testing, surveillance purposes, detecting treatment failure and characterizing outbreaks. The

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primary specimens should be inoculated onto non-selective chocolate agar and selective agar containing antimicrobial agents that inhibit the growth of commensal bacteria and fungi. 3.1.8) PATHOGENICITY: Chlamydiae have the ability to establish long-term associations with host cells. When an infected host cell is starved for various nutrients such as aminoacids (e.g. tryptophan) iron, or vitamins, this has a negative consequence for Chlamydiae since the organism is dependent on the host cell for these nutrients. Long-term cohort studies indicate that approximately 50% of those infected clear within a year, 80% within two years, and 90% within three years. The starved chlamydiae enter a persistent growth state wherein they stop cell division and become morphologically aberrant by increasing in size.[14] Persistent organisms remain viable as they are capable of returning to a normal growth state once conditions in the host cell improve. There is much debate as to whether persistence has in vivo relevance. Many believe that persistent chlamydiae are the cause of chronic chlamydial diseases. Some antibiotics such as -lactams can also induce a persistent-like growth state, which can contribute to the chronicity of chlamydial diseases. 3.1.9) TREATMENT AND PREVENTION: C. trachomatis infection can be effectively cured with antibiotics once it is detected. Current Centers for Disease Control guidelines provide for the following treatments: Azithromycin 1 gram oral as a single dose, or Doxycycline 100 milligrams twice daily for seven to fourteen days. Tetracycline Erythromycin

Non-approved Ciprofloxacin 500 milligrams twice daily for 3 days. (Although this is not an approved method of treatment.) Agents recommended for pregnant women include erythromycin or amoxicillin.

3.1.10) Chlamydia trachomatis And Human Papillomavirus Coinfection: 48 | P a g e

Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection (STI) worldwide.2 Bacterial coinfection by CT in women with a history of HPV infection has been studied as a potential factor that contributes to the development of malignacies and cervical cancer. Some authors3 have suggested that previous CT infection is associated with a high risk of developing the disease. The p16INK4a is a cyclin-dependent kinase inhibitor (CDK) that reduces the cell cycles speed by controlling the expression of E2F transcription factor. High-grade infection by HPV is involved and, HPV integration in the human genome results in overexpression of E6 and E7 viraloncoproteins.4 E7 HPV protein interacts with retinoblastoma protein (pRb) and neutralizes the function of these proteins, resulting in the release of E2F transcription factor of the pRb-E2F complex. E2F accumulation induces p16INK4a activity.5 in the carcinogenesis.[32]

3.2.1) N. gonorrhoeae The genus Neisseria contains a number of species which are normal flora and pathogens of humans and animals. Of these species, the species of human origin--and particularly the pathogenic species, N. gonorrhoeae and N. meningitidis--have been studied extensively in an effort to control the infections they cause.Gonorrhea, caused by N. gonorrhoeae, is one of the most frequently reported infectious diseases in the United States and worldwide. Rapid tests have been developed to identify and distinguish N. gonorrhoeae, from the

commensal Neisseria and related species which are normal flora of the oro- and nasopharynx. Because many rapid tests for the identification of N. gonorrhoeae test for a limited number of characteristics which may be shared by one or more nonpathogenic Neisseria spp., a nongonococcal, commensal Neisseria species may be incorrectly identified as N. gonorrhoeae. Such incorrect identifications may result in serious social and medicolegal consequences for patients and their families. Thus, the primary purpose of these pages is to provide information relating to the accurate identification of N. gonorrhoeae. Descriptions of species in these pages will, for the moment, be limited to those of human origin. Information relating to the identification species of animal origin will include a table of differential characteristics which should be consulted when a gram negative diplococcus is not readily identifiable as a human Neisseria species e.g., an isolate from a wound inflicted by an animal bite. 49 | P a g e

In addition, reference information on the taxonomy, host range, pathogenicity, natural habitat and prevalence of theNeisseria species is included.

3.2.2) Classification : Domain: Bacteria Phylum: Probacteria Class: Beta Probacteria Order: Neisseriales Family: Neisseriaceae Species : Neisseria Gonorrhoeae

3.2.3) Description and significance Neisseria gonorrhoeae is a gram-negative coccus, or bacteria whose overall shape is spherical. It is usually seen in pairs with adjacent sides flattened. The organism is usually found interacellulary in polymorphonuclear leukocytes, or a specific category of white blood cells with varying shapes of nuclei, of the gonorrhea pustular exudates with humans as its only natural host. N. gonorrhoeae is highly efficient in using transferrin-bound iron for in vitro growth. Many strains can also utilize lactoferrin-bound iron. The bacteria bind only human transferrin and lactoferrin. This specificity is thought to be the reason these bacteria are exclusively human pathogens. The bacterium was first discovered in 1879 by a German physician Albert Ludwig Sigesmund.[33] This organism is relatively fragile and is susceptible to temperature changes, drying, UV light, and some other environmental conditions.The recommended procedure for isolating Neisseria gonorrhoeae involves the inoculation of a specimen directly onto a nutritive growth medium that is at room temperature and immediate incubation at 35-37C in an atmosphere of 3 10% added CO2.Strains are inconsistent in their cultural requirements so the media needed for growth and isolation of the organism contain hemoglobin, NAD, yeast extract and other supplements.

3.2.4) Genome structure: Neisseria gonorrhoeae have a circular DNA genome. [7] N. gonorrhoeae strain 1090 genome was sequenced by the University of Oklahoma.The genome length is 2,153,922 nt and contains 2069 genes and 67 structural RNAs. It also has 2002 protein genes. This includes the opacity (Opa) proteins which are responsible for the opaque colony phenotype caused by 50 | P a g e

tight junctions between adjacentNeisseria, and are also responsible for tight adherence to host cells. This organism is also naturally competent for the update of DNA. Neisseria gonorrhoeae can produce one or several Opa proteins. These proteins are subject to phase variation and are usually found on cells from colonies possessing a unique opaque phenotype called O+. At any particular time, the bacterium can express zero, one, or several different Opa proteins, and each strain has 10 or more genes for different Opas. More specifically, during infection, N. gonorrhoeae is like to encounter hydrogen peroxide, which inhibits growth. Since it is an obligate human pathogen, it would not be exposed to typical environmental stress such as UV light, ionizing radiation, or chemical mutagens. The type of DNA damage N. gonorrhoeae would come across is oxidative. [34] N. gonorrhoeae genome contains many genes that are predicted to be involved severeal DNA repair pathyways. Recombinational DNA repair has been studied in N. gonorrhoeae and requires the recA and recX genes, which act with either the RecBCD pathway (recB, recC, and recD genes) or the RecF-like pathway (recO, recQ, recR, and recJ genes). Also, contributing to the recombinational DNA repair pathway is the Holliday junction processing enzymes encoded by recG, ruvA, ruvB, and ruvC. N. gonorrhoeae seems to use both DNA recombinational repair pathways simultaneously. This is in contrast to Escherichia coli, where mutants in the RecF pathway generally show phenotypes only in the context of recBC sbcBC mutations. This leads to the conclusion that recombinational DNA repair is especially important for the repair of damaged DNA in N. gonorrhoeae. During repair of oxidatively damage in E.coli, recA and other reocombinational rpair genes have been shown to be important. E. coli recA is important for both functions in DNA repair and its role in the induction of the SOS response of DNA repair. But because N. gonorrhoeae does not have SOS response, it does not use recA for the repair of oxidatively damaged DNA. Analysis has shown that only recN, a single known DNA repair and recombination gene is upregulated after hydrogen peroxide treatment. It is unclear as to what the exact role of this gene is, but it seems to function in the repair of DNA double strand breaks. In addition, an N. gonorrhoeae recN mutant displays decreased survival to nalidixic acid and hydrogen peroxide, both of which can result in DNA double-strand breaks. Although several gonococcal genes have been identified that protect against oxidative damage, few of them are predicted to function in the repair of DNA. To date, only two genes that are involved in DNA repair and recombination have been found to protect against oxidative damage in N. gonorrhoeae. Both the N. gonorrhoeae recN mutant and a mutant 51 | P a g e

inactivated in priA, which is involved in replication restart, show decreased resistance to oxidative damaging agents. In contrast to E. coli recA, N. gonorrhoeae recA was reported to not protect against oxidative damage caused by H2O2. This suggests that DNA repair and recombination enzymes may differ between N. gonorrhoeae and E. coli in their importance to the repair of oxidatively damaged DNA. RecA, genes of the RecBCD and RecF-like recombination pathways, and genes whose products are involved in Holliday junction processing are all important for mediating repair of oxidative damage. Futhermore, data suggest that these genes are expressed at basal levels sufficient to mediate repair and do not need to be upregulated upon encountering DNA damage in order to function in N. gonorrhoeae. The recent demonstration that N. gonorrhoeae is polyploid suggests that, in the event of chromosomal damage, these additional copies of the chromosome could provide the genetic information present on the damaged copy, perhaps anticipating the necessity of recombinational repair. Therefore, of the many mechanisms of resistance used by N. gonorrhoeae to combat oxidative insult, recombinational DNA repair appears to be one layer of resistance.[35]

3.2.5) Cell structure and metabolism Neisseria gonorrhoeae posses a typical gram negative outer membrane that is composed of proteins, phospholipids, and lipopolysaccharide (LPS). Neisserial LPS is unique in that it has highly-branched basal oligosaccharide structure and the absence of repeating O-antigen subunits. Thus, they are referred to as lipooligosaccharide (LOS). During growth, the bacterium releases outer membrane fragments called "blebs". These contain LOS and may have a role in the pathogenesis if they are distributed during the course of an infection.The bacterium have fimbriae, which is a proteinaceous appendage that is thinner than a flagellum. They play a major role in adherence and extend several micrometers from its cell surface. There are four types of N. gonorrhoeae based on the presences of fimbriae and they are called T1, T2, T3, and T4. In vitro studies show that these piliated cells bind more efficiently to eukaryotic cells than non piliated cells, which suggests that the pilus structure plays an important role in this interaction. [36] N. gonorrhoeae also can move in a jerky fashion across solid surfaces. This type of motility is called twitching, which depends on type IV pili and takes place by a grappling hook mechanism, which is the extension of the pilus, its attachment, and its retraction back into the 52 | P a g e

cell.

Twitching

motility

also

contributes

to

the

formation

of

biofilms.During

growth, Neisseria gonorrhoeae releases soluble fragments of peptidoglycan. These molecules are implicated in the pathogenesis of different forms of gonococcal infection. A major peptidoglycan fragment released by N. gonorrhoeae is identical to the tracheal cytotoxin of Bordetella pertussis and has been shown to kill ciliated fallopian tube cells in organ culture. In the examination of the role of other putative lytic transglycosylases in peptidoglycan-derived cytotoxin (PGCT) production, results suggest that this gonococcal gene (ltgA) encodes a lytic peptidoglycan transglycosylase and that it is responsible for a significant proportion of the PGCT released by N. gonorrhoeae . N. gonorrhoeae genome contains homologues of enzymes involved in PG recycling, and the levels of turnover are consistent with a certain level of recycling occurring in gonococci. It is unknown if N. gonorrhoeae have cytoplasmic proteins for sensing PG fragments; but, this would be a favorable mechanism for controlling cell processes, including autolysis. The presence of two, maybe more enzymes with potentially redundant functions either indicates that gonococci have an elaborate backup system for cell wall processes or may suggest that the enzymes have different functions or are differently regulated or localized. AtlA is encoded in a group of type IV secretion genes in the gonococcal genetic island, and recent evidence suggests that AtlA may have a role in assembly of the type IV secretion system. PGCT is expected to be released during infection, due to the extensive turnover and release of PG fragments in vitro.Although many lytic transglycosylases were characterized in E. coli, the genes for PGCT production have not been previously characterized in bacteria in which PGCT is thought to act in infection.[37] These organisms are aerobic, strongly oxidase-positive, have an oxidative metabolism, are susceptible to drying and are fastidious (growth is inhibited by free fatty acids).

3.2.6) Pathogenesis

Neisseria gonorrhoeae infections are acquired by sexual contact and usually affect the mucous membranes of the urethra in males and the endocervix and urethra in females. The pathogenic mechanism involves the attachment of the bacterium to nonciliated epithelial cells through pili (fimbriae) and the production of lipopolysaccharide endotoxin. Neisseria gonorrhoeae is only found after sexual contact with an infected person (or in the case of infections in the newbord, direct contact). 53 | P a g e

Adherence is mainly done through fimbriae and opa (P.II) protein although nonspecific factors such as surface charge and hydrophobicity may play a role. The bacteria only attach to microvilli of nonciliated columnar epithelial cells and not ciliated cells. After the bacteria attach to the nonciliated epithelial cells of the fallopian tube, they are surrounded by the microvilli that draw them to the surface of the mucosal cell. Then the bacteria enter the epithelial cells by a process called parasite-directed endocytosis. During this process, the membrane of the mucosal cell retracts and pinches off a membrane-bound vacuole that contains the bacteria. The vacuole is transported to the base of the cell, where the bacteria are released by exocytosis into the subepithelial tissue. During infection, bacterial lipooligosaccharide (LOS) and peptidoglycan are released by autolysis of cells. Both bacterial polysaccharides activate the hosts alternative complement pathway, while LOS stimulates the production of tumor necrosis factor (TNF) which causes cell damage. Neutrophils are then attracted to the site and feed on the bacteria. For reasons not known, many gonococci are able to survive inside of the phagocytes.Gonococcal LOS produces mucosal damage in fallopian tube organ cultures and brings about the release of enzymes, such as proteases and phospholipases Thus, gonococcal LOS seems to have an indirect role in mediating tissue damage. Sometimes Neisseria gonorrhoeae can enter the bloodstream causing a Gram-negative bacteremia which may lead to a disseminated bacterial infection. Strains of N. gonorrhoeae that cause disseminated infections are usually resistant to complement and the serum bactericidal reaction. This accounts for their ability to persist in the bacteria infected blood. Gram-negative bacteremias of this kind can be aggravated by the lysing of bacterial cells which may simply liberate soluble LPS.

3.2.7) Virulence Factors

Although it does not produce any exotoxins, Neisseria gonorrhoeae has a wide range of virulence determinants. The first stages of infection, which includes adherence and invasion, are mediated by surface components. The bacterium first attaches to epithelial cells by means of its fimbriae, specifically N-methylphenylalanine pili, with the main subunit PilE. After initial attachment, the bacteria enter a second stage of binding mediated by the outer membrane protein P.II (also known as Opa) which is needed for tight binding and invasion of epithelial cells. Also, P.II from one bacterium will bind to LOS of an adjacent bacterium, which allows for the construction of a small colony that may function similarly to a 54 | P a g e

biofilm. Neisseria gonorrhoeae also produces an IgA1 protease that may take part in the colonization stage.[34] The outer membrane porin of N. gonorrhoeae P.I (also known as Por) is equivalent to the ompC and ompF porins of E. coli. They are involved in the passage of solutes through the outer membrane. However, P.I apparently has a role in virulence that allows the gonococci to survive inside of phagocytes. Purified P.I has also been shown to inhibit the ability of phagocytes to kill ingested bacteria. The lipooligosaccharide (LOS) is thought to be responsible for most of the symptoms of gonorrhea. Gonococcal LOS triggers an intense inflammatory response. The activation of complement, attraction and feeding by phagocytes, and the lysing of the phagocytes themselves, contributes to the purulent discharge. The local production of TNF is thought to be the main cause of damage to the fallopian tubes. In addition, in strains that cause systemic infection, LOS binds sialic acid from the serum forming a microcapsule of sialylated LOS, which allows the gonococci to resist the host immune response and serum bactericidal reaction. Nonsialyated LOS and P.I (Por) on the bacterial surface are known to be effective targets for bactericidal antibodies. However, if antibodies produced against P.III (also known as Rmp) react with their antigenic site on the gonococcal surface, the effect is to block bactericidal antibodies against LOS and P.I and to protect the bacterium from complement-mediated lysis. Neisseria gonorrhoeae also have a well-developed iron acquisition system that allows it to extract iron from its host during growth, which is necessary to support bacterial invasion. The bacterium is able to form two transferrin receptors (Tbp1 and Tbp2) and one lactoferrin receptor (Lbp) in its outer membrane, which are stimulated under low-iron conditions, and are able to directly extract iron from transferrin and lactoferrin. These proteins can also extract iron from heme and hemoglobin. Neeisseria gonorrhoeae usually infects the mucous membranes causing infections such as urethritis, cervicitis, salpingitis, pelvic inflammatory disease, proctitis, conjunctivitis and pharyngitis. 3.2.8) Symptoms In males there is an approximate 2-3 day incubation period after which a purulent discharge from the urethra and dysuria develops. Around 95% of infected males are symptomatic. Rare complications include prostatitis, epididymitis, and periurethral abcesses. In women, Neisseria gonorrhoeae primarily infects the cervix in women. The symptoms of gonorrhea are often mild and most women who are infected do not have symptoms. Even when a woman has symptoms, they can be so non-specific and can be mistaken for a bladder 55 | P a g e

or vaginal infection. Symptoms include vaginal discharge, dysuria, and abdominal pain. Around 10%-20% of infected women develop these complications. In 1%-3% of infected women and a lower percentage of infected men the bacterium disseminates via the blood causing bacteremia and arthritis.[38]

3.2.9) Infection of neisseria:In order to cause disease, most pathogenic bacteria must colonize, grow and persist within their host. The pathogenic Neisseria are an excellent model system to study these processes, since they are highly adapted to life within humans and are able to evade the massive immune response that is typical of neisserial diseases. They also persist as a major problem worldwide despite the availability of effective antibiotic therapies, the World Health Organization estimates that there are ~78 million new cases of infection by N. gonorrhoeae each year.. Part of the success of these bacteria stems from the fact that a significant proportion of infections are asymptomatic, providing the pathogen with an reservoir in which to persist. When disease does occur, the symptoms result from the intensity of the immune response rather than being a direct insult by the bacteria on host cells or tissues. Despite the intensity of the inflammatory response, the specific (memory) response to neisserial infection is typically weak and does not protect against subsequent infections .

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FIG 18 : infection by Neisseria Primary attachment of Neisseria to the apical surface of mucosal epithelial cells is mediated by the bacterial pilus. This filamentous appendage then retracts, pulling the bacterium down onto the host cell membrane. Tight secondary interactions are then mediated by certain variants of the Opa protein family of adhesins. Once attached, the bacteria are able to pass through the the epithelial layer and exit into the subepithelial compartment. They likely tend to remain within this niche, presumably being in contact with cells of the immune system but somehow avoiding triggering a specific immune response. Disseminated gonococcal or meningococcal infections can then occur if the bacterium penetrate across the endothelium and spread through the bloodstream.[39] N eisseria gonorrhoeae infections may present as a broad range of symptoms and can affect urogenital, anorectal, pharyngeal, and conjunctival areas. Severe cases can lead to disseminated gonococcal infections, endocarditis, and meningitis; and in women, to pelvic inflammatory disease (PID). Two methods for detecting N. Gonorrhoeae are culture and nonculture tests. Culture techniques are considered the tests of choice; but nonculture techniques, which are less labor-intensive and are similar in accuracy to cultures, have replaced culture techniques in some instances. newest nonculture technique is the nucleic 57 | P a g e

acid amplification test. This test has good sensitivity (92 to 96 percent) and specificity (94 to 99 percent) compared with cultures.

1.Urogenital Infections : The most common site of N. Gonorrhoeae infection is the urogenital tract. In women it can infect the endocervix and, if an ascending infection develops, it can cause PID. Men may develop urethritis and, occasionally, epididymitis.

2.female infections : In women, common symptoms include odorless vaginal discharge; vaginal bleeding, articularly after intercourse; and dyspareunia. Many women have no symptoms, however. 2 Physical findings in women include cervicitis with mucopurulent drainage from theos. The cervix tends to bleed easily when rubbed with a cotton-tipped swab. Gonorrhea infections do not cause vaginitis, but other concomitant infections may produce vaginal findings. Ten to 20 percent of women with gonorrhoea develop ascending infection that causes acute salpingitis with or without endometritis, also known as PID.2 Presentations may range from no symptoms to severe abdominal pain with a high fever. PID can negatively affect fertility, causing infertility in 15 percent of patients2; 50 percent of patients who have three or more episodes of PID develop infertility.2 The Centers for Disease Control and Prevention (CDC) recommends that physicians aintain a low threshold for diagnosing PID because of significant negative sequelae associated with this infection.2 The CDC currently recommends empiric treatment of PID in women with uterine and adnexaltenderness or cervical motion tenderness if they are at risk of sexually transmitted diseases (STDs) and no other causes can be identified.The CDCs criteria for diagnosing PID. In women with urogenital disease, the nucleic acid amplification test can detect gonorrhea by endocervical or urine sample. Urine samples have a lower sensitivity and, therefore, a higher chance of false negatives than do endocervical samples.

3.Male Infections : Unlike women, men with urogenital infections are usually symptomatic. The normal incubation period is two to six days after exposure. Symptoms include purulent penile discharge and dysuria. The discharge may present at the meatus, which may be erythematous. Discharge may be expressed by milking the penis. N. gonorrhoeae may also cause epididymitis, which can present as unilateral testicular pain without discharge or dysuria. The patient may or may not have a fever. On examination, the epididymis is swollen and tender to palpation. Culture or nonculture techniques can diagnose urogenital gonorrhea in men.1 If the sample is obtained using a urethral swab, the physician should milk the penis 58 | P a g e

first. Nucleic acid amplification tests using urine\ samples provide similar results to that of the urethral swab technique.1

4.Anorectal Infections : Gonorrhea infections in the rectal area are most common in women and in men who have sex with men (MSM). Perianal contamination from a cervical infection or a direct infection from anal intercourse can cause anorectal infections in women. In MSM, the infection is caused by direct exposure through anal intercourse. Most rectal gonococcal infections are subclinical. If present, symptoms can include anal pruritus and mucopurulent discharge, usually with a bowel movement. Rectal pain, tenesmus, and bleeding are more common in MSM. Severe gonococcal rectal infections may be difficult to differentiate from inflammatory bowel disease. Although data suggest that nucleic acid amplification tests can detect rectal gonorrhoea infections,3 the CDC recommends the culture technique for a diagnosis.2 However, one study 4 showed a high prevalence (7 percent) of asymptomatic rectal gonorrhoea in MSM; therefore, it may be beneficial to screen these patients using nucleic acid amplification tests.

5.Pharyngeal Infections : Pharyngeal infections caused by N. Gonorrhoeae usually occur after orogenital exposure;symptoms are mild or absent. On physical examination, the pharynx may be erythematous or have exudates. Anterior cervical lymphadenopathy also may be present. Most cases of pharyngeal infection will spontaneously resolve with no treatment and usually do not cause adverse sequelae. Treatment should be initiated, however, to reduce the potential for spreading the infection.2 The CDC currently recommends cultures to test patients who have suspected pharyngeal gonorrhea.

6.Infections in Children : There are two distinct categories of gonococcal infections in children. During the neonatal period and the first year of life, gonorrhoea infections can cause neonatal conjunctivitis (ophthalmia neonatorum); pharyngitis; rectal infections; and, in rare cases, pneumonia. These infections most commonly develop within two to five days after birth, because the neonate is exposed to infected cervical exudates during delivery. Almost all new gonococcal infections in children older than one year are caused by sexual abuse.

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Treatment : The CDCs treatment guidelines for uncomplicated gonococcal infections are included.2 Despite findings that fluoroquinolones have similar cure rates as ceftriaxone (Rocephin),5 N. gonorrhoeae has become increasingly resistant to fluoroquinolones in some geographic areas. Therefore, the CDC advises against using f luoroquinolones to treat gonorrhea infection in patients who live or may have acquired infection in Asia, the Pacific islands (including Hawaii), and California.2 The CDC recently noted a substantial increase in fluoroquinoloneresistant N. gonorrhoeae in MSM, and it no longer recommends fluoroquinolones as first-line treatment in these patients.6 England, Wales, and Canada also have reported fluoroquinolone-resistant N. gonorrhoeae.7,8 PID can be treated on an outpatient basis if the patient does not meet hospitalization criteria.2 Outpatient treatment may include either of two equally effective oral regimens.2 If parenteral antibiotic therapy is indicated, the preferred therapy is cefotetan (Cefotan), 2 g intravenously every 12 hours, or cefoxitin (Mefoxin), 2 g intravenously every six hours, plus doxycycline (Vibramycin),100 mg orally or intravenously every 12 hours. Doxycycline is best administered orally because intravenous doxycycline can be painful and can adversely affect veins.2 Pharyngeal gonococcal infections are more difficult to treat than urogenital or anorectal infections because few antibiotic regimens can reliably cure this infection. The CDC recommends ceftriaxone in a single 125-mg dose intramuscularly or ciprofloxacin (Cipro) in a single 500mg dose orally.[40]

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4. MATERIALS AND METHODS 4.1 Experimental site:

All the experiments were done in Auroprobe laboratory. it is the ISO 9001:2000 certified molecular diagnostic laboratory. Its offer a wide range of quality assured clinical laboratory test using latest technology. The laboratory is well equipped comprising of reagent and specimen preparation area, bio-safety level 3 facility, PCR, hybrid capture, bactac 460 & COBAS amplicor system. The sample is collected by hospitals & private nursing home from Delhi & NCR region.

4.2) HYBRID CAPTURE TECHNOLOGY FOR DETECTION OF HPV:

Hybrid capture technology (HC), developed by the Digene Corporation, detects nucleic acid targets directly, using signal amplification to provide sensitivity comparable to target amplification methods. Digene has developed two products for the detection of HPV: the first-generation Hybrid Capture Tube (HCT) test and the more recent Hybrid Capture II (HCII) assay. Both assays detect high-risk HPV types. The HCT test detects the following high-risk types (as initially defined by Digene and supported by epidemiological studies): 16, 18, 31, 33, 35, 45, 51, 52, and 56. HCT was granted US FDA approval in May 1995. In March 1999, the US FDA approved Digenes second-generation HPV detection kit (HC II). Four additional viral types were Signal-amplified techniques added to the high-risk category in the HC II test: 39, 58, 59, and 68. The level of detection of the second-generation HC II is rated at 5,000 viral copies per sample, or one picogram of HPV DNA per sample (in contrast to HCT, which detects 10 picograms). To perform the HC assay, cervical or vaginal clinical specimens collected through self-sampling or obtained by a health care provider during a pelvic examinationare combined with an extraction buffer to release and denature the target HPV DNA. The released target DNA then combines with specific RNA probes to create RNA-DNA hybrids, which are captured onto a solid phase by an antibody specific for the hybrids. These captured RNA-DNA hybrids are then tagged with antibody reagents linked to alkaline phosphatase. A chemiluminescent substrate then produces light that is measured on a luminometer in relative light units (RLUs). The amount of light generated is proportional to the amount of target DNA in the original specimen.

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FIG 19: INSTRUMENT AND REAGENTS OF HYBRID CAPTURE ASSAY 4.2.1) Reagents:
Denaturation reagents dilute sodium hydroxide solution (NaoH). Indicator dye- contain sodium azide. Probe diluent- buffered solution with sodium azide contain acetic acid & acrylic acid. HPV probe A- low risk HPV RNA probe coaktail in buffered solution. HPV probe b- high risk HPV RNA probe coaktail in buffered solution. Negative control- carrier DNA in specimen transport medium containing sodium azide. Detection reagent (DR1)- alkaline phosphatase conjugated antibody to DNA-RNA hybrid in buffer solution. Detection reagent (DR2)- chemiluminescent substrate di-oxtane based compound . Wash buffer contains sodium azide.

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4.2.2) Equipment:
Dml 2000 chemiluminometer Rotary shaker Wash apparatus Specimen collection tube rack Pipette Vortex mixer Water bath Single channel micropipette Timer

4.2.3) Accessories:
Microtube Microtube racks Plate sealers Extralong pipette tip for removal of specimen Digene sample conversion kit Disposable reagent reservoir Absorbent paper Microwell plate Test tube rack

4.2.4) Preparation Of Reagent:

COCKTAIL- RNA probe + diluents RNA probe per sample 1.2 l Diluent per sample 30 l WASH BUFFER- Dilute 100ml wash buffer concentrated with2.9ml of double distilled water and mix well.

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4.2.5) REAGENT STORAGE:

1. Store the kit at 2-8C 2. Denaturation reagent is stable for three months when stored at 2-8C 3. Wash buffer is stable for 3 months at 2-25C

4.3) METHOD: 1. Sample (1ml transport medium) containing double stranded DNA which helps
a. to maintain the integrity of the DNA, NC and PC containing 50 l carrier b. DNA. 2. Pipette 500 l of denaturation reagent in the sample and add 25 l of denaturetion reagent in NC and PC.(add denaturation reagent in 2:1) and vertex it. 3. Incubate sample along with NC and PC in waterbath at 65C for 1 hour. 4. Transfer 75 l sample in microtubes. 5. Add 25 l of probe mix in each 75 l sample along with NC and PC. 6. Again incubate tubes in water bath in 65C for 10 min. 7. Change the precoated antibody microplate and shake at1100rpm for 1 hour on rotary shaker. 8. Add 75 l of detection reagent 1(DR1) into each well of microplate . 9. Cover microwell plate with plate sealer. 10. Shake it at 1100 rpm for 1 hour on rotary shaker. 11. Wash all the microwells 6 times with wash buffer. 12. Add 75 l of detection reagent2 (DR2) into each well of microplate. Microplate should turn yellow colour. 13. Read captured microplate on luminometer. 14. Get the Relative light unit (RLU) value of all the samples including the PC and NC. 15. Get the RLU/ cut off value by dividing the RLU values of the samples by the RLU values of the PC. 16. If the value is <0.99, then the sample would be negative. 17. If the value is >0.99, then the sample would be positive.

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4.4) Protocol:
1 ml. Sample (NaoH & dye) with in tubes

Add just half of it denaturation solution vortex Incubate at 65C for 45 to 60 min.

Transfer 75l of sample in microtubes including PC and NC

Then transfer cocktail (mix) 25l in to each tubes of sample.

Incubate it 65C for 40 min.

Change than precoating antibody microplate shake 1100 rpm for 1 hr. Add 75l detection reagent1 (DR1) Shake at 1100 rpm for 1 hr

6 times wash with wash buffer

Add 75l detection reagent2 (DR2)

Read capture microplate on Luminometer

Validate assay and interpret specimen result

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4.5) The basic steps of the Hybrid Capture assay are as follows:
1. Release Of Nucleic Acids

Clinical specimens are combined with a base solution which disrupts the virus or bacteria and releases target DNA. No special specimen preparation is necessary.

2. Hybridise RNA Probe with Target DNA

Target DNA combines with specific RNA probes creating RNA:DNA hybrids.

3.

Capture

Hybrids

Multiple RNA:DNA hybrids are captured onto a solid phase coated with universal capture antibodies specific for RNA:DNA hybrids.

4.

Label

for

Detection

Captured RNA:DNA hybrids are detected with multiple antibodies conjugated to alkaline phosphatase. Resulting signal can be amplified at least 3000-fold.

5. Detect, Read and Interpret Results

The bound alkaline phosphatase is detected with a chemiluminescent dioxetane substrate. Upon cleavage by alkaline phosphatase, the substrate produces light that is measured on a luminometer in Relative Light Units (RLUs).

4.6) Characteristics of Hybrid Capture II Technology:

Hybrid Capture 2 (hc2) technology serves as the platform for QIAGENs nucleic acid hybridization assay for detection of human papillomavirus (HPV), Chlamydia trachomatis (CT), and Neisseria gonorrhoea (NG). This diagnostic method allows rapid, standardized 66 | P a g e

testing of genetic material of the infectious agents in virtually any laboratory setting. It employs specific RNA probes, hybridization, antibody capture, and signal amplification that utilizes qualitative chemiluminescent detection.

The digene HPV Test, using Hybrid Capture 2 technology, provides an accurate, costeffective, user-friendly method by achieving reliable detection without the need for dedicated and isolated lab space, sophisticated laboratory expertise, or concern related to inhibition and contamination.

4.6.1) Benefits of Hybrid Capture:

Same-day, objective results using a micro-plate test format High-throughput automation for handling increasing test volumes Minimal required specimen preparation allowing samples to be processed quickly and efficiently Less complex workflow than target amplification methods Reduced risk of cross-over contamination due to signal amplification instead of target amplification Extensive clinical validation data available

4.7) Relative Light Unit (RLU) and Result Interpretation:

A chemiluminescent substrate then produces light that is measured on a luminometer in relative light units (RLUs). The amount of light generated is proportional to the amount of target DNA in the original specimen. An RLU measurement equal to or greater than the Cutoff Value (CO) indicates the presence of high-risk HPV DNA sequences in the specimen. An RLU measurement less than the Cutoff Value indicates the absence of the specific highrisk HPV DNA sequences tested or HPV DNA levels below the detection limit of the assay. 1. Specimens with RLU/CO ratios 1.0 are considered positive. 2. Specimens with RLU/CO ratios < 1.0 are considered negative or non detected for the 13 HPV types tested. High-risk HPV DNA sequences are either absent or the HPV Solution DNA levels are below the detection limit of the assay. 3. When testing Preserve Solution specimens, if the RLU/CO ratio of a specimen is 1.0 and < 2.5, the specimen must be retested. If the initial retest result is positive ( 1.0 RLU/CO), the specimen can be reported as positive and no further retesting needs to be completed. However, if the first retest result is negative (< 1.0), then a second retest (third result) needs

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to be completed to generate a final result. The result of the second retest is considered the final result and is to be reported . 4. Because this assay only detects high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, other low-risk HPV types may be present in the specimen.

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5).DETECTION METHOD FOR HSV & CT/NG

5.1 Sample collection The various clinical samples for our experiment were collected from different hospitals of Delhi, and samples were analyzed. The DNA were isolated from the samples by using the ZR Viral DNA Kit.

5.2) Polymerase Chain Reaction (PCR) was done on each sample which involves following steps: DNA Isolation PCR Set up and Amplification Detection of amplified PCR products can be assayed.

5.3) DNA Isolation Reagents for DNA isolation It include following reagent Proteinase K Lysis Buffer Wash buffer Elution buffer Ethanol Autoclaved ultra pure water (MQ water with 18 mega ohms resistance)

5.4) Equipments and accessories for DNA isolation Biosafety cabinet (Class 2) Centrifuge (10,000 g, rotor for 50 ml, 1.5 ml and 0.2 ml micro tubes), Vortex Shaker Homogenizer Aerosol barrier tips (20 l, 200 l and 1000 l). Pipettes (20 l, 200 l and 1000 l). Latex gloves Micro centrifuge tubes(1.5 ml)

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5.4 ) Storage of Each Content of the DNA isolation Kit All contents of the Kit except Proteinase K are stable at room temperature (20-250C) for one year. Spin Columns: Storage (dry) at room temperature - up to lyear without showing any reduction in performance. Collection Tubes :-Storage at room temperature - forever. Proteinase K:- Storage at 40C - upto 2-3 months after reconstitution. - Storage at -200C- upto 1 year, but should avoid repeated freeze-thawing. - Dividing the solution into aliquots and storage at -200 C is recommended. 5.5) Protocol of DNA isolation 200l Homogenite Sample.

Add 200l Lysis Buffer (B3) +20l Proteinase k. Vortex(10-20 sec) Then Incubate at 550 C on Heating Block for 1 hours.

Add 200l Lysis Buffer-2. Vortex Incubate at 70o C on Heating Block for 10 to 15 minutes.

Add 200l Absolute Ethanol. Vortex Transfer the Sample (680 l) in Column Tubes with the Collection Tubes.

Centrifuge at 11,000 rpm for 2 minutes.

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Discard the Collection Tubes and Transfer the Collumn Tubes in new Collection Tubes.

Add 500l Wash Buffer 1 . Centrifuge at 11,000rpm for 1 minutes

Discard the Collection Tubes and Transfer the Column Tubes in a new Collection Tubes.

Add 600l Wash Buffer- 2

Discard the Supernatent and dry wash at 11,000 rpm for 2 minutes.

Then transfer the Column in MCT (Microcentrifuge Tubes).

Add 60l Pre warm Elution Buffer.

Incubate at Room Temperature for 5 minutes. Then Centrifuge at 14,000 rpm for 2 minutes.

DNA Elute. After DNA isolation, 12.5 l master mixes is added in the eluted DNA.

5.6) PCR BASED DETECTION OF HSV, CT/NG The polymerase chain reaction (PCR) is an in-vitro, enzymatic and exponential amplification of the target DNA sequence under controlled thermal conditions. PCR is performed in a microprocessor controlled machine, the Thermo cycler, which provides controlled temperature conditions under an automatic monitoring system.

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When the reaction is allowed to take place under the most appropriate conditions, it is rapid, sensitive and specific. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase are key components to enable selective and repeated amplification. The result is the exponential accumulation of the specific nucleic acid sequence from a single starting copy, easily detected by various analysis systems- Gel electrophoresis, ELISA, etc. The process of PCR is divided into 2 steps - : 1. 2. Preparation of Master Mix Amplification phase

5.7) Master Mix preparation Composition of master mix

SN Reagent concentration) (with working Final volume in 50ul

1. 2. 3. 4. 5. 6. 7. 8. 9.

Buffer dNTPs P1 P2 P3 P4 Taq poly. MQW Total volume

5 5 1 1 1 1 0.25 10.75 25.00

5.8) Procedure and instructions for making Master Mix:

1. Carry all the reagents for master mix preparation into AREA I, in a cool box. 2. Always wear sterile powder free gloves while preparing the master mix as powder can adversely affect the activity of Taq polymerase.

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3. Thaw all the reagents before use. 4. Mix all the reagents well before aliquoting , otherwise changes in the concentration will disturb the accuracy of quantity. 5. Companies provide Taq Polymerase with its specific buffer and MgCl2. ( It is important to use Taq Polymerase, Bufffer and MgCl2 of the same lot no., for the optimum activity of Taq polymerase). 6. Before proceeding to make the MMX , ensure that all the working reagents are available. 7.set up of PCR reaction 5.9)Set Up Of PCR Reaction: PCR setup has to be performed with following the steps given below; 1. l Master Mix aliquot PCR tubes and allow it to thaw completely if stored in 20oC. 2. Mix with gentle finger tapping and spine shortly to settling down MMX to the bottom of PCR tube. 3. Mark on the tube based on sequence of DNA sample and positive control. 4. Add 25 l of isolated DNA template and negative control to the MMX. The final volume would become 50l for reaction. 5. Pipette up and down to mix DNA template with MMX. 6. Spin briefly for a second in spin win to bring down reaction mix to the bottom of PCR tube. 7. Switch on Thermal cycler for five minutes before start reaction for auto calibration. 8. Keep all tubes into the thermal cycler block. 9. Set the program for amplification as follows for 35 cycles.

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Temperature (C) 94oC 94oC 55oC 72oC 72oC 04oC

Time (in min) 6min. Denaturation 1min. 1.5min, 40 Cycles. 1.5min. 7min. 8 min.

Table.2 Sequence of temperature in PCR set up

10. After completion of amplification keep PCR amplified product in refrigerator at 4oC until the detection of amplified product.

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5.10) Electrophoresis And Detection Of Amplified Product Electrophore and resolve the amplified product on 1.6 % agarose gel to detect the band of interest. Follow the steps mentioned below for electrophoresis. Preparation of 1.6% Agarose Gel 1. Sealed the Gel casting tray with tape and placed on a horizontal surface. The well forming comb has to be positioned parallel to the tray. 2. Measure 125 ml 1X TAE Buffer with the help of measuring cylinder of 250ml capacity and pour into 250ml conical flask. 3. Weigh 2.0 gram agarose on electronic balance and add into conical flask containing 125 ml of 1X TAE buffer. 4. Keep in microwave oven to boil till the complete dissolution of agarose added to the flask. 5. Take out the conical flask and allow it to cool down up to the 40-50oC then add 5.5l of Ethidium-bromide (Conc. 0.5mg/ml) and mix thoroughly. 6. Pour boiled agarose into sealed casting tray without creating any air bubbles then allow it to solidify at room temperature. 7. Pour 40-50ml 1X TAE buffer on the solidified gel and pull out comb upwardly without disturbing wells. 8. Place the gel in electrophoresis tank and fill the tank with 1X TAE buffer to cover the gels to a depth of 1mm.

5.11) Preparation of Gel Loading Buffer: To make 20 ml gel loading buffer, weigh 8 grams sucrose (40%, w/v), 50 mg Bromophenol blue (0.25% w/v) and dissolve in 15 ml Milli Q water. Make up the volume 20 ml with Milli Q water with the help of 25 ml measuring cylinder.

5.12) Preparation of DNA Ladder: Readymade - Prepare as recommended by the commercial supplier. 1. A known molecular weight marker (100bp DNA Ladder) should be loaded in the first well. 2. 20 l of PCR product is loaded into each well along with 4 l of gel loading buffer. 3. Positive control and a negative control are loaded along with the samples. 75 | P a g e

4. Electrophoresis is carried out at 100-150 Volt (5 to 8 V/cm for 20 cm gel) until the bands in the molecular weight marker are resolved. Then the gel is examined under UV light (302nm) on ULTRA LUM Electronic UV transilluminator gel documentation system for the presence of 240 base pair PCR product and photographed. Caution: Ethidium Bromide is a powerful mutagen and toxic. Wear gloves when working and a mask when weighing the powder.UV light is harmful to eyes so UV protection shield should be used always.

Appearance of 147 bp specific band indicates the presence of HSV- I and 227 bp band indicates the presence of HSV-II. Appearance of 463 bp specific band indicates the presence of CT Appearance of 260 bp specific band indicates the presence of NG

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Figure : Agarose gel showing positive and negative samples of HSV and NG

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6) RESULT AND DISCUSSION

The aim of this prospective study was to identify the positivity rate of HPV,HSV,CT/NG in different age group and sex of patients by using hybrid capture technology and PCR approach in a selected population of Delhi (NCR)or to check co-infection between these diseases. Samples were collected from different collection centers and for these diseases many different kind of sample are collected given below. The total number of samples collected was 100 for each disease in a period of 4. The Sample for the HPV is mainly cervical brushing is or endocervical swap,epithelial cells,scrapping of warts ,fluid of warts collected in sampler tubes.the sample is collected by an instrument known as speculum. The sample is then stored transported in atube containing viral medium and stored in 4 C.The sample for CT is either urine or uretheral swabs which is to be collected in a CT sampler tube. Analysis of PCR results is based on the presence or absence of specific bands of amplified DNA in agarose gel (2%).

6.1) DATA ANALYSIS: 4) Table: shows the characteristics of patients showing age group distribution of HPV.
Age years) 20-30 30-40 40-50 >50 Total samples 49 25 17 9 100 groups(in Total no. of sample Total no. of positive Positive cases 37 15 6 4 62 %) 75.5 60 35.3 44.4 62 cases (in

The results show that in patients highest positive cases(37%) in the age group 20-30 years and lowest(4%) in the age group >52years. 15% of the cervical cancer was also found to be present in the age group 30-40 years of HPV infection. Only women with a positive hpv result required immediate reffered to colposcopy.

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5) Table : PREVELANCE RATE FOR HSV

S.No.

Specimen Types

Total

No.

of No. specimens +ve -ve 84 2 2 1

of Prevalence

specimens

1. 2. 3. 4.

CSF Blood Cervical Swab Corneal scrapping

90 3 3 1

6 1 1 0

6.66% 33.33% 33.33% 0

5.

Serum Total

3 100

0 8

3 0

0 8%

Total number patients positive for HSV= 8 Total Number of patients= 100 Detection limit = Total number patients positive for HSV 100 Total Number of patients = 8% Positivity Rate Positive, 8, 8%

Positive negative

negative, 92, 92%

Fig. 15: Positivity Rate.

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In our study, PCR, the positivity rate of PCR for the detection of HSV I & II DNA in CSF were 6%. The 6 out of 90 clinical samples was found positive for HSV. With HSV diagnosis, accurate information about diagnosis, prognosis, and treatment options, i.e., early episodic treatment or suppressive prophylaxis in cases with frequent recurrences, is possible. There were large number of negative results, these PCR results may be explained by the fact that the DNA in the investigated samples may be around the level of detection and may be unevenly distributed.

6) Table : Rate of occurrence of HSV in male and female among positive patients. Sex Total no. of positive No. patients Male Female 8 8 of positive % positivity

samples 6 2 75% 25%

Positivity rate among male and female

Female, 2, 25%

Male Female

Male, 6, 75%

Fig. 16: Rate of occurrence of HSV in male and female among positive patients. This showed that females are less infected than the males according to our study.

Methods useful in the diagnosis of HSV infection in some clinical situations, are timeconsuming and have a low sensitivity when a low amount of viable viruses are expected. Nucleic acids amplification methods may be a useful diagnostic approach in these situations. 80 | P a g e

We want to emphasize the great advantage of using a reaction mixture previously prepared and kept frozen until use with same performances as fresh prepared one. This simplifies to a great extent the procedure and makes this technique feasible to clinical laboratories.

7) Table : Determination of Positivity rate in age groups of HSV

S.No.

Age groups

No. of positive samples

1. 2. 3. 4. 5.

0-10 10-20 20-30 30-40 40-50

1 1 4 2 0

The samples we collected were of age between 3 years to 80 years. They were grouped into 5 age groups. There was one female sample with HSV positive result, between 30-40 years of age, this showed that the HSV 2 infection is prevalent in them as compared to males. A male child of 3.6 years was found positive.

8) TABLE : PREVELANCE RATE FOR CT

S.No.

Specimen Types

Total

No.

of No. specimens +ve -ve 1

of Prevelance

specimens

1.

VAGINAL SWAB

66.6%

2. 3.

URINE CERVICAL BRUSH

18 22

1 3

17 19

5.55% 13.6%

4.

PELVIC FLUID

50%

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5.

CERVICAL SMEAR Total 50

0%

43

14%

9) Table : Determination of Positivity rate in age groups of CT

Age groups(in years) 20-30 30-40 40-50 >50 Total samples

Total no. of sample

Total no. of positive cases

Positive cases (in %) 16 13.3 12.5 0 14%

25 15 8 2 50

4 2 1 0 7

10) TABLE : PREVALENCE RATE FOR NG

S.No.

Specimen Types

Total No. Of No. Specimens

Of Prevalence

Specimens +Ve -Ve 5 17 4 2 4 39 16.6 22.7 0 33.3 40 22.5

1. 2. 3. 4. 5.

Vaginal Swab Urine Cervical Swab Pus Cervical Brushes Total

6 22 4 3 5 40

1 5 0 1 2 9

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11) Table : Determination of Positivity rate in age groups of NG

Age groups(in years) 20-30 30-40 40-50 >50 Total samples

Total no. of sample

Total no. of positive cases

Positive cases (in %) 18.75 27.7 20 0 22.5

16 18 5 1 40

3 5 1 0 9

The results show that in patients of CT/NG highest positive are present in the age group 2035 years and lowest in the age group >52years.

12) Analysis Of Positive Samples For HPV & HSV Viral Disease :-

Viral Diseases HPV HSV

Total no. of samples 100 100

No. of positive results 62 8

Positivity rate(%) 62% 8%

Chart analysis of positivity rate in Viral samples

100 80 60 40 20 0 HPV HSV

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13) Analysis Of Positive Samples For CT/NG Bacterial Disease :-

50 45 40 35 30 25 20 15 10 5 0 CT NG

14) Table - Age wise distribution of positive samples in HSV and HPV:

Serial no 1. 2. 3. 4. 5. 6.

Age (yrs)

HPV positive 0 0 37 15 6 4

HSV positive

0-10 11-20 21-30 31-40 41-50 51-60

1 1 4 2 0 0

Positivity rate of HPV(%) 0 0 75.5 60 35.3 44.4

Positivity rate of HSV(%) 33.3 50 7.4 9.5 0 0

The results show that in patients highest positive cases in the age group 20-30 years and the prevalence of HPV is higher than HSV and CT/NG. Lowest prevalence found in case of CT

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CO-INFECTION IN STIs: We only find co-infection between HPV and Chlamydia trachomais (CT) patients. A total of 100 patients were analyzed, among which 62% were positive for HPV-DNA and 14 % for CT-DNA. Only 2 women with positive CT-DNA were also positive for HPV-DNA.

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7). CONCLUSION: I did my research on four STIs. This study was used to see the prevalence of HSV,HPV,CT/NG by diagnosing various clinical samples collected from the NCR regions, India and to examine the parameters associated with the collection. Human papillomavirus is a virus that affects both Men and Women. The human papilloma viruses are small DNA tumor viruses that belong to the family of papoviridae. The incidence of cervical carcinoma worldwide is estimated to be as high as 400,000 diagnoses per year. More than 90 percent of high grade cervical dysplasia and invasive cervical cancers have been associated with 0 to 15 high risk HPV viral types. Women with an equivocal Pap smear or low grade cervical dysplasia are advised to have HPV test. Those women having HPV result negative can be safely rapture to pap smear routine screening. There risk of cancer is negligible. Only women with a positive HPV result required immediate reference for colposcopy. Our results show that the highest positive cases in the age group <40 years and lowest positive cases in the age group >50years. 15% of the cervical cancer was also found to be present in the age group 30-40 years of HPV infection. In case of HSV, CT/NG the amplification was proper which enabled us to come to a proper conclusion. These results strongly suggest that PCR could be used for a rapid complementary diagnosis of HSV. A prospective study to evaluate the PCR results, clinical evolution, and treatment is imperative to corroborate the real value of PCR in diagnosis and how it could help the clinicians' approach. The number of CSF (cerebrospinal fluid) sample, collected were maximum which also showed high positivity rate as compared to the other samples collected. One of the female sample with cervical swab was found to positive for HSV, so optimal management of genital herpes and strategies to prevent its transmission include antiviral therapy to suppress viral shedding as well as education and counseling regarding the natural history of HSV, the risk of sexual and perinatal transmission, and preventive methods. This depends on accurate detection of infected persons.We found that the humans in the age groups of 15-30 were most affected, and the our study showed that males were more affected than females. These results suggest the need for integrating HSV- 1 & 2 treatment in the syndromic management of the disease. In case of Chlamydia trachomatis (CT) and Neisseria Gonorrhoeae (NG) is the most common bacterial sexually transmitted infection (STI) worldwide. The results show that in 86 | P a g e

patients of CT/NG highest positive are present in the age group 20-35 years and lowest in the age group >52years. We find co-infection in case of HPV and CT only.Although HPV is a necessary cause of cervical cancer, it is not a sufficient cause. Thus, other cofactors are necessary for progression from cervical HPV infection to cancer. Long-term use of hormonal contraceptives, high parity, tobacco smoking, and co-infection with HIV have been identified as established cofactors; co-infection with Chlamydia trachomatis (CT) and herpes simplex virus type-2 (HSV-2), Neisseria Gonorrhoeae, immunosuppression, and certain dietary deficiencies are other probable cofactors. Sometimes these diseases can occur simultaneously.

The result shows that only 2 of the women is coinfected by both HPV and CT. Most of women with age group 25-30 are suffering for both HPV and CT and presence of HPV is more than HSV and CT/NG.

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of Medical Microbiology and Immunology, University of Wisconsin Medical School, American Society for Microbiology, 2002 38. NCBI: Neisseria Gonorrhoeae, Accessed August 23, 2007 39. Gray-Owen, S.D. et al. (2001) Neisseria. In: Principles of Bacterial Pathogenesis. Editor E.A. Groisman. Academic Press, San Diego, CA. 40. KARL E. MILLER, M.D., University of Tennessee College of Medicine, Chattanooga Unit, Chattanooga, Tennessee Diagnosis and Treatment of Neisseria gonorrhoeae Infections

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