Laboratory detection of bacterial pathogens and clinical and laboratory response of syndromic management in patients with cervical discharge: A retrospective study

Background: Cervical discharge as part of cervicitis and pelvic inflammatory disease is a cause of significant morbidity in sexually active women worldwide. Non-gonococcal and non- chlamydial bacterial pathogens are becoming more prevalent. Aims: This study aims to determine bacterial pathogens causing cervical discharge using culture and/or polymerase chain reaction and assess the clinical and laboratory response to the conventional syndromic kit regimen established by the World Health Organisation. Methods: A retrospective review of records of women with cervical discharge over one year period. Culture and/or polymerase chain reaction results of endocervical swabs of various bacterial pathogens at baseline and after four weeks of treatment with syndromic kit regimen were recorded. Results: A total of 70 case records were reviewed for clinical details, out of which results of bacterial culture and polymerase chain reaction were available for 67 cases. Infectious aetiology was found in 30 (44.7%) patients with Ureaplasma species being the most common organism isolated on culture (18, 26.8%) and polymerase chain reaction (25, 37.3%), respectively. Polymerase chain reaction for Chlamydia trachomatis and Mycoplasma hominis was positive in ten (14.9%) and four (6%) cases, respectively. None of the patients showed positive culture for Neisseria gonorrhoeae. Coinfection was seen in eight (11.9%) patients with the majority showing Chlamydia trachomatis and Ureaplasma spp. coinfection (five patients). Forty one cases (58.5%) received tab. cefixime 400 mg and tab. azithromycin one gram stat (kit 1), while 29 cases (43.3%) received tab. cefixime 400 mg stat, tab. metronidazole 400 mg and cap. doxycycline 100 mg, both twice daily for 14 days (kit 6). Minimal to no clinical improvement with treatment was seen in 14 out of 32 cases (44%) at the end of four weeks with the conventional kit regimen. Post-treatment culture and/or polymerase chain reaction were positive in nine out of 28 cases (32.1%) with Ureaplasma spp. being the most common. Limitations: Retrospective study design, small sample size and fewer cases with follow-up data were the main limitations. Conclusion: Ureaplasma spp. was the most common infectious cause of cervical discharge in our patients. Treatment given as part of syndromic management led to a clinical and microbiological response in around half and two-third cases, respectively.

Corynebacterium striatum: An emerging nosocomial skin and soft-tissue pathogen

The genus Corynebacterium is composed of Gram-positive, aerobic, non-motile, non-spore-forming bacilli that are widely distributed throughout the environment. They are usually found as commensals on the skin and are often considered as mere contaminants when isolated from clinical samples. We describe a patient with skin and soft-tissue infections due to Corynebacterium striatum following exploratory laparotomy identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The clinical importance and pathogenic potential of Corynebacterium species, especially C. striatum, cannot be underestimated. This report is a reminder to physicians of the possible pathogenicity of non-diphtherial Corynebacteria.

Chlamydia trachomatis serovar G infection in a bisexual male with urethritis.

We report a case of Chlamydia trachomatis serovar G urogenital tract infection in a 33-year-old human immunodefi ciency virus-1 (HIV-1) seropositive Indian bisexual male. This case highlights the emergence of a new serovar in India. The patient was tested positive for C. trachomatis by both cryptic plasmid and ompA gene polymerase chain reaction (PCR). On further characterization using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and ompA gene sequencing, the strain was found to be C. trachomatis serovar G. His spouse was also found to be infected with C. trachomatis serovar G. Phylogenetic analysis was performed on the clinical isolates obtained from both partners and were found to be identical to the isolates available in GenBank. The sexual network could not be traced further. Detection of a new genotype suggests importation of a new strain into the population probably by sexual contact with a person from a geographical area where the strain is common. Identifying circulating genotypes in the community can assist in developing strategies for improved sexually transmitted disease control.

Genital Mycoplasma and Chlamydia trachomatis infections in patients with genital tract infections attending a tertiary care hospital of North India.

Limited data are available on the prevalence of genital mycoplasmas and Chlamydia trachomatis (CT) among Indian patients with genital tract infections. The objectives of the study were to determine the prevalence of Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), and CT in patients with genital tract infections. The antimicrobial susceptibilities of UU and MH were also assessed. Endocervical swabs/urethral swabs and first void urine samples of patients (n = 164) were collected. UU and MH were detected by culture and multiplex polymerase chain reaction (PCR). MG and CT were identified by PCR. Ureaplasma isolates were further biotyped and serotyped. Antimicrobial susceptibility was done by microbroth dilution method. UU, MH, MG, and CT were detected in 15.2%, 5.4%, 1.2%, and 6% patients, respectively. Ureaplasma parvum serovar 3/14 was the most prevalent. All isolates of UU and MH were uniformly susceptible to doxycycline and josamycin. Routine screening for these pathogens and antimicrobial susceptibility testing is warranted to prevent sequel of infections and formulate treatment guidelines.

Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India.

Background & objectives: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. tThe in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. Methods: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA) and enzyme immunoassay (EIA) Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. Results: C. trachomatis infection was found in 13.5 per cent (27/200) infertile women by in-house real time-PCR, 11.5 per cent (23/200) by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200) by DFA and 6.5 per cent (7/200) by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. Interpretation & conclusions: iIn-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C. trachomatis infection in women attending an infertility clinic. In an effort to prevent Chlamydia infection associated infertility, we recommend screening of women with infertility due to C. trachomatis infection by in-house molecular method as a cost-effective solution in resource limited settings.

Prospective study estimating healthcare associated infections in a paediatric hemato-oncology unit of a tertiary care hospital in north India.

Background & objectives: Healthcare associated infections (HAIs) are responsible for morbidity and mortality among immunocompromised and critically ill patients. We undertook this study to estimate the burden of HAIs in the paediatric cancer patients in a tertiary care hospital in north India. Methods: This prospective, observational study, based on active surveillance for a period of 11 months was undertaken in a 4-bedded isolated, cubicle for paediatric cancer patients. Patients who stayed in the cubicle for ≥48 h, were followed prospectively for the development of HAIs. Results: Of the 138 patients, 13 developed 14 episodes of HAIs during the study period. Patient-days calculated were 1273 days. Crude infection rate (CIR) and incidence density (ID) of all HAIs were 9.4/100 patients and 11/1000 patient-days, respectively. Of the 14 episodes of HAIs, seven (50%) were of blood stream infections (HA-BSI), five (36%) of pneumonia (HAP) and two (14%) urinary tract infections (HA-UTI). The CIRs of HA-BSI, HAP and HA-UTI were 5.1, 3.6 and 1.4/100 patients, respectively. The corresponding IDs were 5.5, 3.9 and 1.6/1000 patient-days, respectively. Mean length of stay was significantly higher in patients who developed an HAI [13.8 (range 7-30), median (Interquartile range) 12 (11-14)] vs 7.5 days [range 2-28, median (interquartile range) 7 (5-9); P<0.0001]. Also mortality was significantly higher in patients who developed an HAI [23% (3/13) vs 3% (4/125), P<0.05]. Interpretation & conclusions: The incidence of HAIs in the paediatric cancer patients in the study was 11/1000 patient days, of which HA-BSIs were the commonest. HAIs were associated with an increase in morbidity and mortality amongst this high risk patient population.

0.5% Proparacain hydrochloride for clear corneal phacoemulsification in patients with co-morbid conditions.

Genital tuberculosis is a common cause of female infertility in India. But, it is important to screen for other agents like Chlamydia trachomatis and genital Mycoplasmas as well to avoid persistence of infection and its long-term sequelae. Timely diagnosis of these infections using nucleic acid amplifi cation tests and institution of appropriate therapy will improve the conception rates in infertile women. We report a case of co-infection of Mycoplasma genitalium and Chlamydia trachomatis in an infertile female patient with genital tuberculosis. The infections were diagnosed using polymerase chain reaction, and the patient responded to a combination of antituberculosis therapy and 1 g single-dose Azithromycin.

Co-infections with Ureaplasma parvum, Mycoplasma hominis and Chlamydia trachomatis in a human immunodeficiency virus positive woman with vaginal discharge.

A 30-year-old human immunodeficiency virus (HIV)-1 infected woman presented with vaginal discharge and associated vulval irritation. The vaginal swabs tested positive for Ureaplasma parvum and Mycoplasma hominis by both culture and polymerase chain reaction (PCR). The specimen also tested positive for Chlamydia trachomatis deoxyribonucleic acid (DNA) by cryptic plasmid and omp1 gene PCR assays. The patient was successfully treated with azithromycin based on the antibiotic susceptibility testing results of U. parvum and M. hominis by microbroth dilution. Since sexually transmitted infections enhance the transmission of HIV, HIV-positive patients should be screened routinely for these pathogens.

Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods.

Background & objectives: Bartonella henselae is a fastidious gram-negative bacterium usually causing self limiting infections in immunocompetent individuals but often causes potentially life threatening infection, such as bacillary angiomatosis in immunocompromised patients. Both diagnosis of infections and research into molecular mechanisms of pathogenesis have been hindered by lack of appropriate and reliable diagnostic techniques. We undertook this study to standardize methods to characterize B. henselae in clinical samples to diagnose Bartonella infection correctly. Methods: B. henselae ATCC 49882 strain was procured from American type culture collection, USA. This strain was revived and maintained in the laboratory, and identification and characterization of this strain was done by conventional and molecular techniques, which included culture on various media, staining by different methods including electron microscopy, biochemical analysis by conventional methods and API, polymerase chain reaction (PCR) for amplification of citrate synthase gene followed by restriction fragment length polymorphism (RFLP). Results: This organism was biochemically inert due to slow growth and generated unique identification code with API. The amplification of the citrate-synthase gene with primers yielded a 381 bp product followed by specific RFLP profile for B. henselae. Interpretation & conclusions: Bartonella is fastidious and fragile organism and should be handled carefully. Extra effort and careful observation are required to isolate and characterize this organism.

Ureaplasma serovars & their antimicrobial susceptibility in patients of infertility & genital tract infections.

Background & objectives: Ureaplasmas have been implicated in a variety of clinical conditions. However, only certain serovars of ureaplasmas are disease associated. Only a few classes of antimicrobial agents are available for the treatment of mycoplasmal infections in humans. Increase of resistance of genital mycoplasmas to antimicrobials has been reported worldwide. The aim of the present study was to determine the occurrence of Ureaplasma serovars in patients with infertility and genital tract infections with polymerase chain reaction (PCR)–based serotyping. The antimicrobial susceptibilities of Ureaplasma spp. and Mycoplasma hominis were also assessed to determine the most suitable treatment strategy. Methods: Sexually active adults (n=147) with symptoms of genital tract infections and 115 infertile women were enrolled. Endocervical swabs from women and urethral swabs from men were subjected to culture and multiplex PCR for detection of genital mycoplasmas. Serotyping of Ureaplasma was done by PCR and antimicrobial susceptibility to doxycycline, azithromycin, josamycin and ofloxacin was done by microbroth dilution method. Results: Ureaplasma was detected in 25.8 per cent patients with genital tract infections and 20.8 per cent in infertile women. Serovar 3/14 was the most frequent isolate followed by serovar 1 and serovar 6. The majority of Ureaplasma isolates were susceptible to doxycycline (91%) and josamycin (86%) followed by ofloxacin (77%) and azithromycin (71%). All the isolates of M. hominis were uniformly susceptible to doxycycline, josamycin and ofloxacin. Interpretation & conclusions: The predominance of Ureaplasma serovar 3/14 suggests their possible pathogenic role in genital tract infections and infertility. For empirical treatment, doxycycline could be the drug of choice for genital mycoplasmas.

Genital mycoplasma & Chlamydia trachomatis infections in treatment naïve HIV-1 infected adults.

Background & objectives: Sexually transmitted infections (STIs) enhance the transmission of human immunodeficiency virus (HIV). Thus, screening for STIs is a routine component of primary HIV care. There are limited data for selective screening guidelines for genital mycoplasmas and Chlamydia trachomatis in HIV-infected adults. The aim of the present study was to determine the frequency of genital infections with Ureaplasma spp., Mycoplasma hominis, M. genitalium and C. trachomatis in treatment naïve asymptomatic HIV-1 - infected adults and study their association with CD4+ T-cell count. Methods: First-void urine samples were collected from 100 treatment-naïve HIV-1-infected adults and 50 healthy volunteers. C. trachomatis and M. genitalium were detected by polymerase chain reaction (PCR). Ureaplasma spp. and M. hominis were detected by both culture and PCR. Circulating CD4+ cell counts of HIV-1-infected patients were determined from peripheral blood by flow-cytometry. Results: C. trachomatis was detected in 7 per cent of HIV-1-infected adults compared to none in control population. Ureaplasma spp. and M. hominis showed infection rates of 6 and 1 per cent in the HIV group and 2 and 0 per cent in the control group, respectively. None of the individuals from the patient and control groups was tested positive for M. genitalium. A significant association was found between CD4 cell count and detection of C. trachomatis in HIV-infected adults (P = 0.01). Interpretation & conclusions: Screening of HIV-infected individuals for C. trachomatis infection could be recommended as a routine component of HIV care. The role of mycoplasmas as co-pathogens of the genitourinary tract in HIV-1 infected patients seems to be unlikely. Further longitudinal studies need to be done to confirm these findings.

Sero diagnosis of Legionella infection in community acquired pneumonia.

Background & objectives: Legionella pneumophila has been increasingly recognized as an emerging pathogen responsible for community acquired pneumonia (CAP) worldwide. In India, the actual burden is not known. The present study was thus undertaken to see the presence of Legionella infection in patients with community acquired pneumonia admitted in a tertiary care centre in north India. Methods: Both children and adults (n=113) with symptoms of pneumonia were included in the study. Clinical samples (blood, urine, nasopharyngeal aspirates, bronchoalveolar lavage, sputum, etc.) were collected and subjected to culture and other tests. Enzyme linked immunosorbent assay (ELISA) was done by commercial kits for all the three classes of immunoglobulins (IgG, IgM & IgA). Urinary antigen was also detected using commercial kits. Culture was performed on 51 respiratory tract fluid samples. Serum samples of 44 healthy controls were also screened for the presence of anti-legionella antibodies (IgG, IgM & IgA). Results: Thirty one of the 113 cases (27.43%) were serologically positive. Anti-legionella IgG, IgM and IgA antibodies were positive in 7.96, 15.92 and 11.50 per cent patients respectively. In controls, seropositivity was 9.09 (4/44). IgA was positive in 3 and IgM, IgG combined in one. Antigenuria detection by Microwell ELISA kit showed 17.69 per cent positivity. Four antigenuria positive patients were also serologically positive; of these two patients were positive for IgM, hence considered as confirmed cases of Legionella infection. None of the sample was culture positive. Interpretation & conclusions: Combination of serology and antigenuria detection may be a valuable tool for the diagnosis of Legionella infection in absence of culture positivity. In order to evaluate the actual burden of Legionella in community acquired pneumonia, further studies with larger samples need to be done.

Outcome in childhood guillain-barré syndrome.

To assess the outcome of children diagnosed with Guillain-Barré syndrome (GBS), followed up for a median duration of 25 months. Methods. Tertiary center, prospective follow up of children with GBS enrolled from Dec 2003 through Sep 2006. Functional recovery was determined at 12 months and later using Hughes scale (0-6). Clinical, electrophysiological variables were compared between the good outcome (grade 0/1) and bad outcome groups (died or functional grade >1). Results. Among 52 children with a median age of five yr there was male preponderance (75.4%). Mortality during acute phase was 11.5% (6/52). Among the survivors long term data was obtainable in 40 of the 46 children. At one year follow up 87.5% children had fully recovered or had minimal symptoms, beyond one year this rose to 95%. Only 2 among 40 had significant symptoms at last follow up (1 grade-2 and 1 grade-3). Factors significantly associated with poor outcome were: need for artificial ventilation, inexitable nerves on nerve conduction testing and delayed independent walking. Conclusion. Children needing ventilation have the worst short-term prognosis. However, irrespective of severity during acute phase, good long-term recovery can be expected in most children.

Tigecycline susceptibility report from an Indian tertiary care hospital.

Background & objectives: Treatment of serious life threatening infections due to multi-drug resistant pathogens presents a difficult challenge due to the limited therapeutic options. Therefore, we studied the in vitro susceptibility of tigecycline, a new glycylcycline with promising broad spectrum of activity against Gram positive and Gram negative bacteria at a tertiary care hospital in north India. Methods: A total of 75 multi-drug resistant isolates of methicillin resistant Staphylococcus aureus (21), vancomycin resistant enterococci (14), vancomycin resistant Streptococcus spp. (3), extended spectrum β lactamase producing Gram negative bacteria (11) and multi-resistant Acinetobacter spp. (26) were tested for tigecycline susceptibility by the E- test and disc diffusion methods. An additional 83 multi-resistant Gram negative clinical isolates were screened by disc diffusion method alone. Results: All the isolates of MRSA, VRE, vancomycin resistant Streptococcus spp. and ESBL producing enteric bacteria were sensitive to tigecycline by the E-test and disc diffusion methods. However, only 42 per cent of Acinetobacter spp. were found to be sensitive to tigecycline by the E-test method. Interpretation & conclusions: In conclusion, tigecycline was found to be highly effective against Gram- positive bacteria and Gram-negative members of Enterobacteriaceae, but a high prevalence of resistance in members of Acinetobacter spp. is worrisome.

Pulmonary involvement in leptospirosis: a diagnostic enigma.

Leptospirosis is spirochetal zoonosis. It is endemic in India and pulmonary manifestations of this disease remain under-recognised. We describe a case with leptospirosis with pulmonary manifestations and highlight the importance of its early and accurate diagnosis. The case also highlights the importance of considering leptospirosis in the differential diagnosis in patients presenting with febrile illness and pulmonary manifestations.

Changing pattern of Clostridium difficile associated diarrhoea in a tertiary care hospital: a 5 year retrospective study.

BACKGROUND & OBJECTIVE: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. METHODS: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP). RESULTS: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives. INTERPRETATION & CONCLUSION: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.

Clinical significance of airways colonization with Ureaplasma urealyticum in premature (<34 wk) neonates.

BACKGROUND & OBJECTIVE: Ureaplasma urealyticum has been implicated in various neonatal morbidities in preterm infants. Its association with chronic lung disease (CLD) remains controversial. The aim of this prospective study was to investigate colonization of U. urealyticum in preterm infants (with gestational age <34 wk) and to evaluate the relationship between U. urealyticum colonization and neonatal morbidity including CLD. METHODS: U. urealyticum was cultured from nasopharyngeal or endotracheal aspirates collected within 24 h of birth from infant <or=34 wk gestation weighing <1800 g admitted to a Neonatal Intensive Care Unit of a tertiary care hospital in north India, and PCR was performed on the DNA extracted from these samples. RESULTS: Twenty per cent of the study infants were colonized with U. urealyticum. The mean gestational age of the infants in the colonized group was less than that of non colonized infants (P<0.05). The peripheral total leukocyte counts and mortality rate were higher in infants with U. urealyticum colonization than in non-colonized infants (P<0.05). There was no significant difference between the colonized and non colonized groups with regard to the antenatal use of steroids, sex, cause of respiratory distress, use of surfactant, duration of ventilation. INTERPRETATION & CONCLUSION: None of the 20 babies colonized with U. urealyticum developed CLD as compared with two (2.5%) of the non colonized group. Colonization of the airways with U. urealyticum had no significant role in development of CLD in Indian preterm infants.