Simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis by PCR in genitourinary specimens from men and women attending an STD clinic.

Neisseria gonorrhoeae and Chlamydia trachomatis are the two most common bacterial sexually transmitted infections that manifest primarily as urethritis in males and endocervicitis in females, though the infection may be asymptomatic especially in women. Since complications may occur in untreated symptomatic and asymptomatic infected individuals, early diagnosis and treatment of infected individuals is required to prevent severe sequelae and spread of these diseases. Recently molecular amplification assays like Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) have been found to be highly sensitive and specific methods for detection of N. gonorrhoeae and C. trachonmatis not only in urethral and cervical specimens but also in urine. The objective of this study was to screen male and female Sexually Transmitted Disease (STD) clinic attenders, with and without symptoms suggestive of urethritis and cervicitis for presence of N. gonorrhoeae and C. trachomatis using a multiplex PCR based assay, to compare its performance with culture for N. gonorrhoeae and Direct Fluorescent Antibody (DFA) staining for C. trachomatis and also to compare the efficacy of PCR test performed on urine and genital swab specimens collected from this high risk group. Genital specimens and urine was collected from STD clinic attenders. N. gonorrhoeae and C. trachomatis was detected in genital specimens by culture and DFA respectively. Multiplex PCR was used to detect N. gonorrhoeae and C. trachomatis infection in both genital and urine specimens. Among men with urethritis, N. gonorrhoeae was detected in 70% by culture and 77% by PCR, while C. trachomatis as detected in 7.5% by DFA and 17.5% by PCR. Among females with endocervicitis, N. gonorrhoeae was detected in 7.7% by culture and 30.7% by PCR, while C. trachomatis was detected in 7.7% by DFA and in 15.4% by PCR. None of the asymptomatic males were positive for N. gonorrhoeae and C. trachomatis by conventional methods, while 43.9% were positive for N. gonorrhoeae and 7.5% for C. trachomatis by PCR. Fifty per cent of asymptomatic women were positive for C. trachomatis by PCR alone. We encountered PCR positive but culture/DFA negative results and also PCR negative but culture/DFA positive results. In view of this a single PCR test cannot be used for diagnosis and treatment of N. gonorrhoeae and C. trachomatis infection unless confirmed by a second test.

Seroprevalence of HSV1 and HSV2 infections in family planning clinic attenders.

Few studies from India documented seroprevalence of HSV 1 and 2 infection in different population groups. We determined HSV 1 and 2 seroprevalence in a cohort of adults 16-40 year of age, attending the family planning clinic. For the overall study population, 63% were seropositives, 33.3% for HSV 1 alone. 16.6% for HSV 2 and 13.3% had mixed infection. By the statistical analysis, the mean age difference between the two sexes for either infection was not significant. HSV-2 seroprevalence was associated with an increasing age. Men were more likely than women to be seropositive for HSV2. More studies from India are required to coroborate our findings.

Pediatric HIV infection.

OBJECTIVE: The present cross sectional study was undertaken to study clinical profile of HIV infection in children in Northern India. METHODS: 64 children from newborn to eighteen years, presenting for confirmation of diagnosis of HIV infection or monitoring of CD4-CD8 counts in confirmed cases, were evaluated. Children were categorized as per CDC classification of Pediatric HIV. The diagnosis was confirmed by serological tests or PCR assay. CD4-CD8 counts were done by FACS Count. RESULTS: Majority of the children were between 18 months to 5 years. Adolescents comprised 24% of the case. 51.5% children were infected through the mode of mother to child transmission. 39% of the case was transfusion-mediated. Unsafe medical injections probably contributed to 6.2% and heterosexual promiscuity led to 3.1% cases. Clubbing, not described in Indian studies so far, was seen in 9.3% cases. CONCLUSIONS: HIV infection is a chronic childhood disease extending into adolescence, and contaminated blood and unsafe medical injections are still important routes of HIV transmission in India.

A cross sectional serological study of the co-infection of hepatitis b virus, hepatitis c virus and human immunodeficiency virus amongst a cohort of idus at Delhi.

A cross-sectional study was undertaken to find out co-prevalence of various infectious markers like Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV), Hepatitis C virus (HCV) and Syphilis infection amongst a cohort of injecting drug users (IDUs) in the city of Delhi. A total of 246 IDUs were enrolled during the 3 months period of the study. The results revealed a high prevalence of the viral markers studied i.e., HBV-39.59%, HCV-36.45%, HIV-36.99% and Syphilis-6.09%. A single marker infection was detected amongst 9.14% for HBV, 8.37% for HCV, 4.87% for HIV and 0.83% for Syphilis in samples tested for multiple markers. All the four markers could be detected in 1.76%. Amongst 11.16% and 27.9% of these samples, three and two markers respectively could be detected. The study revealed the problem of IV drug use and high prevalence of infectious markers including HIV in certain populations of Delhi and emphasizes the need for relevant interventions in these localised pockets.

Cytokine profile in human immunodeficiency virus positive patients with and without tuberculosis.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with a profound dysregulation of the immune system and alterations in the cytokine profile. Tuberculosis, a common opportunistic infection in HIV positive patients, leads to further immune suppression and a faster progression of the disease. This study was conducted to assess and compare the cytokine profiles in HIV positive subjects with and without pulmonary tuberculosis. METHODS: Twenty HIV positive patients and 20 controls were evaluated after being divided into four groups of 10 each--group 1--HIV positive patients without tuberculosis, group 2--HIV positive patients with pulmonary tuberculosis, group 3--healthy adults and group 4--HIV negative patients with pulmonary tuberculosis. After clinical evaluation, the CD4 cell counts and the cytokine profiles--interleukins (IL)-2, 4, 6, 10, 12, interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) were studied in all the subjects at the start and after three months of antituberculosis therapy. RESULTS: the CD4 cell counts were significantly lower in groups 1 and 2 compared to groups 3 (p < 0.01) and the counts in group 2 were significantly lower than in group 1 in both at baseline (p = 0.0076) and at three months (p = 0.089). The levels of the type 1 cytokines (IL-2, IL-12 and IFN gamma were significantly lower in patients in groups 1 and 2 compared to group 3 (p < 0.001 for both). The levels in group 2 were lower than in group 1 (p < 0.001). The type 2 cytokines (IL-4, IL-6, IL-10 and TNF alpha) were higher in group 1 and 2 compared to group 3, though statistically significant for IL-6 in group 1 (p < 0.05) and for IL-4, IL-6 and TNF alpha in group 2. A highly significant (p < 0.0001) positive correlation between CD4 cell counts and type 1 cytokines and a highly significant (p < 0.0001) negative correlation between CD4 cell counts and type 2 cytokines was observed. CONCLUSIONS: A decline in the type 1 and a rise in type 2 cytokines was observed in HIV infection. Patients in group 2 had the lowest CD4 cell counts, lowest levels of type 1 cytokines and highest levels of type 2 cytokines denoting maximum immunosuppression.

Serodiagnosis of extra pulmonary tuberculosis using A-60 antigen.

The diagnosis of extrapulmonary tuberculosis including neurotuberculosis is difficult because of the low yield of culture positivity for Mycobacterium tuberculosis (M. tb). Serodiagnosis has emerged as a useful aid to the diagnosis of extrapulmonary tuberculosis. The utility and efficacy of detection of antimycobacterial antibodies to A-60 antigen in serum and/or cerebrospinal fluid (CSF) was analysed in 100 patients-neurotuberculosis-72, abdominal tuberculosis-12 and others-16. The overall positivity rate for the test was 75%. The positivity rate of the test in serum and/or CSF was 79.2% (57 of 72) in neurotuberculosis and 62.5% (10 of 16) for other forms of extrapulmonary tuberculosis. The positivity rate for antimycobacterial antibodies was higher for patients with tubercular meningitis -94.7%. To conclude, testing for antimycobacterial antibodies to A-60 antigen is a useful adjunct in the diagnosis of extrapulmonary tuberculosis especially neurotuberculosis.

Synergistic immunosuppression by candida in HIV infection: a cytokine based analysis.

Candida is a common opportunistic pathogen in HIV infection and is regarded a signal infection for progression to AIDS. Cytokine imbalances between Th1/Th2 groups have been described in both candida and HIV infections. A study was undertaken to assess the role of candida in furthering immunosuppression in HIV infection based on cytokine levels and CD4 cell counts. 30 Indian subjects were enrolled; 10 HIV positive patients with and 10 without mucosal candidiasis and 10 age matched controls. Th1 cytokines; interleukin (IL) 2, IL 12 and interferon (IFN) gamma, Th2 cytokines; IL 4, IL 6, IL 10 and tumor necrosis factor (TNF) alpha with CD 4 cell counts were estimated using ELISA in all subjects. CD4 cell counts were reduced in both patient groups as compared to controls; significantly more in patients with both HIV and candida infections. There was a decrease in Th1 cytokine levels in all patients; lower levels of Th1 cytokines were seen in patients with both infections. Among the Th2 cytokines, there was a significant increase in the levels of IL 6, IL 10 and TNF alpha in both patient groups; IL 10 and TNF alpha values were significantly raised in patients with dual HIV and candida infections as compared to the other patients. There was no difference in IL 4 values across the subject groups. A positive correlation between CD4 cell counts and Th1 cytokine levels and a negative correlation with Th2 cytokines were noted; these were stronger in patients with both HIV and candidiasis. Thus, there was a Th1/Th2 cytokine imbalance with CD4 cell count reduction in all HIV infected patients, which was more pronounced in patients with both infections. It can be concluded that, owing to the depressed CD4 cell count and Th1 response and increased Th2 cytokines in patients with both candidiasis and HIV as compared to patients with only HIV candidiasis may have a synergistic immunosuppressive effect with HIV in patients with dual infections.

Tumour necrosis factor-alpha and interleukin-2 in pus aspirate and blood in patients with amoebic liver abscess.

OBJECTIVES: Cell mediated immunity (CMI), cytokines and humoral immunity have been implicated in the pathogenesis of invasive amoebiasis. METHODS: The role of cytokines--tumour necrosis factor-alpha (TNF-alpha) and interleukin 2 (IL-2) in blood and pus aspirate was studied in 20 patients of amoebic liver abscess (ALA), before and after treatment and 10 controls. RESULTS: The mean TNF-alpha levels (pg/ml) in the controls and before treatment in the patients in serum and pus were 24.3 +/- 11.6, 28 +/- 14.5 and 161.2 +/- 81.3 (p < 0.002) respectively. The mean IL-2 levels (pg/ml) in the controls, serum and pus aspirate in the patients prior to treatment were 10.3 +/- 8.5, 39.2 +/- 26.1 and 117.0 +/- 65.9 respectively. The levels in the patients after therapy, increased to 47 +/- 25.7 (p < 0.001) and 134 +/- 59.4 (p < 0.01). CONCLUSIONS: The higher levels of TNF-alpha and IL-2 in the pus aspirate compared to blood pre treatment, supports the role of locally released cytokines in the target organ i.e. liver in amoebiasis. The rise in values observed after therapy are indicative of increased macrophage activity due to CMI occurring late in the course of the disease which may contribute to disease limitation and localisation in amoebiasis. The study suggests that locally released cytokines play an important role in the pathogenesis of ALA.

Acid fast staining versus ELISA for detection of Cryptosporidium in stool.

A prospective study was undertaken to compare the efficacy of ELISA in detection of Cryptosporidium Specific Antigens (CSA) in stool specimens of patients attending various OPDs in a Delhi Hospital. A total of 216 consecutive faecal specimens were examined microscopically after modified acid fast staining. An ELISA was also performed using ELI-WFLL CRYPTO detection kit following the manufacturer's instructions for detection of CSA in stool specimens. Taking microscopy as the gold standard ELISA was found to be 100% sensitive and 99.07% specific in detection of Cryptosporidium spp. The test is easy to perform and interpret.

Giardia lamblia: in-vitro sensitivity to some chemotherapeutic agents.

The susceptibility of Giardia lamblia to eight chemotherapeutic agents was studied in vitro. The criteria of viability of the parasite was the ability of the parasite to attach to the coverslip surface in situ, and to multiply in drug free media on subcultures. The giardicidal and 50% inhibitory concentrations of each drug were measured. The activity of metronidazole in vitro was found to be similar to that of mepacrine, whilst that of chloroquine was inferior. The sensitivity of G. lamblia to mepacrine and chloroquine is markedly greater than that reported for Entamoeba histolytica.

Immunodiagnosis of human hydatid disease.

A study was undertaken to evaluate the efficacy of Enzyme Linked Immunosorbent Assay using locally prepared antigens for immunodiagnosis of human hydatid disease. A total of 90 cases clinically suspected to be suffering from hydatid disease and 100 controls matched for age and sex were included in the study. Two types of ELISA were performed on detected specific antihydatid antibodies belonging to IgG/IgM/IgA classes and other type detected IgE class of antibodies. Antigen prepared from the human hydatid fluid was found to be unsuitable for diagnosis as it contained host proteins i.e. IgG. Sheep hydatid fluid obtained from the fertile hydatid cyst was used to prepare and standardize the antigen. ELISA test to detect anti hydatid antibodies belonging to either IgG, IgM and or IgA was found to be highly specific (98 per cent) in surgically confirmed hydatid disease and was negative in all the controls. The results of the study indicate that ELISA along with casoni test may provide the best results in diagnosis of hydatid disease.

Clinical spectrum of chronic liver disease in north Indian children.

Recent advances in serodiagnosis of hepatotropic viruses have revolutionized the approach to diagnosis and understanding of chronic liver disease (CLD). There are few studies on CLD in children from India. The present study was planned to define the clinical spectrum of CLD in children, its histopathology and seroepidemiology. Forty children with clinical features satisfying the criteria for diagnosing chronic liver disease were studied. All underwent routine laboratory investigations, liver function tests and ultrasound scan of the abdomen. Liver biopsy, upper GI endoscopy and other special investigations were done wherever indicated. The most common presenting features were jaundice (70%), fever (67%), and abdominal distention (60%). On examination hepatomegaly and icterus (80% each) and splenomegaly (67%) were the commonest findings. Serum transaminases were raised in 62.5% of children while prothrombin time was prolonged in 75% patients. Oesophageal and/or gastric varices were seen in 13 out of 29 patients subjected to upper GI endoscopy. Hepatitis B surface antigen (HbsAg) was positive in 5 children (12.5%) while 3 (7.5%) tested positive for anti HCV antibody. The commonest histopathological diagnosis was infantile cholangiopathy (20%) followed by cryptogenic cirrhosis and idiopathic chronic active hepatitis (17.5% each). The study suggests that the incidence of chronic hepatitis B and C is rather low in childhood. However larger and longer studies are required to delineate the exact incidence of these conditions in childhood and their progression in adolescence and early adulthood.

Laboratory techniques for diagnosis of chlamydial infections of the eye.

Giemsa and fluorescence antibody (FA) staining were used to diagnose patients clinically suspected to be suffering from trachoma. A total of 52 controls i.e. individuals with refractive errors and no clinical trachoma and 173 cases suffering from different stages of trachoma were studied. FA was found to be 2.52 times more sensitive in confirming the presence of Chlamydia trachomatis compared to Giemsa staining. 28/52 (53.8%) and 4/52 (7.,69%) controls were also positive by FA and Giemsa staining, respectively, indicating sub-clinical infection without symptoms. Post treatment staining with both methods revealed that clinical cure of trachoma did not necessarily mean the absence of Chlamydia trachomatis in the conjunctival smears. As a corollary it can be deduced that mere presence of Chlamydia trachomatis in conjunctival epithelial cells may not cause clinical trachoma, certain host factors (local immunity etc.) may play an important role in clinical disease.

Determinants of symptomatic giardiasis in childhood.

Giardia has been frequently implicated as a causative agent for acute as well as chronic diarrheal diseases in children. The present study was aimed at exploring the determinants of manifestations of Giardiasis in childhood, in relation to various host and parasite related factors. A total of 200 children with acute (100), chronic (50) or without (50) diarrhea in last 15 days were recruited for the study and evaluated with regards to nutritional status, serum immunoglobulins, secretory IgA levels, presence of Giardia in stool/duodenal aspirate/duodenal biopsy specimen and for associated infections. Lysates from acute giardiasis cases were further studied for zymodeme (banding) pattern. After correlation of all investigations, humoral immune defect in the host was found to be the major determinant of whether the Giardial infestation would be symptomatic or not, while associated bacterial infections and zymodeme patterns were not found to be important in determining the pathogenicity or presentation of giardiasis.

A study of immune profile in human hydatid diseases.

A clinico haematological and immunological study was undertaken in 90 patients clinically suspected to be suffering from hydatid disease over a period of 1 year. The parameters studied included age of presentation, site of cyst localisation, haematological profile, total immunoglobins of different classes (IgG, IgM, IgA & IgE) and complement component C3, rosette forming lymphocytes, blast cell formation in response to phytohaemagglutinin (PHA-P) and concanavalin A (Con-A) and casoni test using standard methods. Twenty two out of 90 (22.44%) clinically suspected patients were surgically confirmed as hydatid disease cases. Hydatid disease occured in all age groups. Youngest case was 8 years and oldest 70 years. In 17/22 cases the cyst localised in the liver followed by lungs (3) neck (1) and kidney (1). Majority of patients (63.65%) belonged to blood group B. The mean total leucocyte and eosinophill counts were raised significantly (p < 0.001 and p < 0.05, respectively) in confirmed patients. The mean ESR value was raised in hydatid patients though, not significantly (P > 0.08). All the four classes of immunoglobulins viz. IgG, IgA, IgM, IgE and complement C3 were significantly raised in patients of hydatid disease compared to controls (P < 0.001, P < 0.002, p < 0.01 and p < 0.01, P < 0.02 respectively). The percentage of lymphocytes in peripheral blood in hydatid patients was reduced though, not significantly (P > 0.2). The absolute lymphocyte count was raised and mean percentage of T cells was reduced in patients with hydatid disease (P < 0.001 and P < 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the immune and nutritional status of the host in childhood diarrhoea due to cryptosporidium.

Cryptosporidium oocysts were detected microscopically in the concentrated faecal smears (stained by modified kinyoun's acid fast stain) in 13 out of 100 (13 per cent) cases of acute diarrhoea (AD < 2 weeks duration), 7 out of 50 (14 per cent) cases of chronic diarrhoea (CD > 2 weeks duration) and none in 50 age matched controls. The grades of malnutrition of the cases and controls were calculated by the weight for age criteria and the immune status assessed by the levels of serum immunoglobulins and SIgA in duodenal fluids. Malnutrition was observed in 6 out of 13 cases (46.1 per cent) in acute and 6 out of 7 cases (85.71 per cent) in chronic cryptosporidial diarrhoeas. There was no significant statistical difference (P > 0.05) in serum immunoglobulins and SIgA levels in chronic cryptosporidiosis. SIgA was significantly reduced (P > 0.05) in cases of acute cryptosporidiosis. Cryptosporidium is an important cause of symptomatic infection in apparently immunocompetent children not having been detected in a single non-diarrhoeal control. Further a low SIgA could contribute to acute symptomatic cryptosporidiosis by favouring colonization with the parasite.

Kinetics of specific IgM in patients of hepatic amoebiasis.

Entamoeba histolytica (EH) specific IgM was measured in 54 patients with diagnosed amoebic liver abscess (ALA), 13 with non-suppurative hepatic amoebiasis (NSHA) and 50 controls. The mean levels of EH specific IgM, estimated by ELISA were significantly raised in patients of invasive amoebiasis (both ALA and NSHA) compared to controls (P less than 0.05). EH specific IgG was also raised in both groups of patients. Follow up of patients with ALA showed a significant decline (P less than 0.05) in the specific IgM levels three months after treatment while the specific IgG antibodies persisted in high titres (1:160). Only four patients of NSHA could be followed up and all showed a decline in specific IgM levels. Raised specific serum IgM seems to be an indicator of active (invasive) amoebiasis.

Colonic immunity in patients and amoebic liver abscess.

Secretory immunoglobulin A (S-IgA), coproantibody titre (antiamoebic) and IgA, IgG, IgM immunocytes in rectal mucosa were studied in 13 patients with amoebic liver abscess (ALA) prior to and 4-6 weeks after completion of antiamoebic therapy. Ten asymptomatic Entamoeba histolytica cyst passers and 17 healthy age and sex matched volunteers served as controls. Fecal S-IgA levels and counts of IgA bearing immunocytes in mucosa were significantly higher in patients with ALA and cyst passers as compared to healthy controls and showed a significant fall after treatment. Fecal antiamoebic antibodies were high in cyst passers and in cases of ALA after treatment. Raised levels of S-IgA and IgA class immunocyte counts probably indicate a local mucosal immune response directed at containing the infection.