Pan-India influenza-like illness (ILI) and Severe acute respiratory infection (SARI) surveillance: epidemiological, clinical and genomic analysis.

Background: Over time, COVID-19 testing has significantly declined across the world. However, it is critical to monitor the virus through surveillance. In late 2020, WHO released interim guidance advising the use of the existing Global Influenza Surveillance and Response System (GISRS) for the integrated surveillance of influenza and SARS-CoV-2. Methods: In July 2021, we initiated a pan-India integrated surveillance for influenza and SARS-CoV-2 through the geographically representative network of Virus Research and Diagnostic Laboratories (VRDLs) across 26 hospital and laboratory sites and 70 community sites. A total of 34,260 cases of influenza-like illness (ILI) and Severe acute respiratory infection (SARI) were enrolled from 4 July 2021 to 31 October 2022. Findings: Influenza A(H3) and B/Victoria dominated during 2021 monsoon season while A(H1N1)pdm09 dominated during 2022 monsoon season. The SARS-CoV-2 "variants of concern" (VoC) Delta and Omicron predominated in 2021 and 2022, respectively. Increased proportion of SARI was seen in extremes of age: 90% cases in < 1 year; 68% in 1 to 5 years and 61% in ≥ 8 years age group. Approximately 40.7% of enrolled cases only partially fulfilled WHO ILI and SARI case definitions. Influenza- and SARS-CoV-2-infected comorbid patients had higher risks of hospitalization, ICU admission, and oxygen requirement. Interpretation: The results depicted the varying strains and transmission dynamics of influenza and SARS-CoV-2 viruses over time, thus emphasizing the need to continue and expand surveillance across countries for improved decision making. The study also describes important information related to clinical outcomes of ILI and SARI patients and highlights the need to review existing WHO ILI and SARI case definitions.

Mutation Analysis of SARS-CoV-2 Variants Isolated from Symptomatic Cases from Andhra Pradesh, India.

There has been a continuous evolution in the SARS-CoV-2 genome; therefore, it is necessary to monitor the shifts in the SARS-CoV-2 variants. This study aimed to detect various SARS-CoV-2 variants circulating in the state of Andhra Pradesh, India. The study attempted to sequence the complete S-gene of SARS-CoV-2 of 104 clinical samples using Sanger's method to analyze and compare the mutations with the global prevalence. The method standardized in this study was able to amplify the complete length of the S-gene (3822 bp). The resulting nucleotide and amino acid mutations were analyzed and compared with the local and global SARS-CoV-2 databases using Nextclade and GISAID tools. The Delta variant was the most common variant reported in the present study, followed by the Omicron variant. A variant name was not assigned to thirteen samples using the Nextclade tool. There were sixty-nine types of amino acid substitutions reported (excluding private mutations) throughout the spike gene. The T95I mutation was observed predominantly in Delta variants (15/38), followed by Kappa (3/8) and Omicron (1/31). Nearly all Alpha and Omicron lineages had the N501Y substitution; Q493R was observed only in the Omicron lineage; and other mutations (L445, F486, and S494) were not observed in the present study. Most of these mutations found in the Omicron variant are located near the furin cleavage site, which may play a role in the virulence, pathogenicity, and transmission of the virus. Phylogenetic analysis showed that the 104 complete CDS of SARS-CoV-2 belonged to different phylogenetic clades like 20A, 20B, 20I (Alpha), 21A (Delta), 21B (Kappa), 21I (Delta), 21J (Delta), and 21L (Omicron).

HPV genotype prevalence in Indian women with cervical disease and estimation of the potential impact of HPV vaccines on prevention of cervical cancer.

PURPOSE: Human papillomavirus (HPV) causes genital and oropharyngeal cancers worldwide. There are significant gaps exist in the data on HPV genotype prevalence in this part of the country. HPV vaccination is one of the best preventive methods available currently. HPV genotyping plays an important role in the selection of appropriate vaccines and monitoring vaccine efficacy and coverage. The present study aimed to determine the HPV genotype prevalence and to estimate the potential impact of HPV vaccines on invasive cervical cancer. MATERIALS AND METHODS: A total of 204 cervical biopsy samples collected from symptomatic women were subjected to an in-house designed and standardised nested multiplex PCR (NM-PCR) assay. The NM-PCR was designed to detect 38 Mucosal HPV types as a pooled result and genotyping of 15 HPV types. Further, the HPV genotype data was used to estimate the HPV vaccine bivalent, quadrivalent and nonavalent impact on the population using a mathematical formula. RESULTS: Out of 204 samples 188 were subjected to HPV-nested PCR. A total of 163 (86.7%) samples were positive for at least one HPV type. Multiple genotypes were identified in 30% of samples processed. HPV-16 (85.3%) was the most frequently detected genotype followed by HPV-18 (13.5%) and HPV-33 (11.0%). Other genotypes were observed less frequently. Based on the HPV prevalence observed in the study a mathematical model estimated the efficacy of bivalent, quadrivalent and nonavalent vaccines were 76.1%, 76.7%, and 91.1% (average) respectively. CONCLUSIONS: HPV-16 was the most prevalent (>85%) genotype detected in this study. Multiple infections observed in 30% of samples were quite high as compared to the majority of national, and global reference (15-25.4%) data. The Mathematical model showed that a nonavalent vaccine would give better protection.

A randomised controlled trial of high-flow nasal oxygen versus non-rebreathing oxygen face mask therapy in acute hypoxaemic respiratory failure.

Background and Aims: A major limitation to providing oxygen therapy by high flow nasal oxygen (HFNO) delivery devices is its availability and therefore as an alternative many clinicians use a standard non rebreathing face mask (NRBM) in order to oxygenate their patients where low-flow nasal oxygen or simple facemask oxygen is not providing adequate respiratory support to achieve the target peripheral oxygen saturation (SpO2). We aimed to determine the clinical effectiveness of HFNO versus NRBM in terms of improving patient outcome among patients admitted to our intensive care unit (ICU) during coronavirus disease-2019 (COVID-19) outbreak. Methods: In this prospective open labelled study, 122 COVID-19 patients presenting with acute hypoxaemic respiratory failure (AHRF) were randomised to receive either HFNO or NRBM to achieve the target SpO2. The primary clinical outcome measured was device failure rate and secondary outcome was all-cause 28-day mortality rate. Results: The device failure rate was significantly higher in HFNO group (39% versus 21%, P = 0.030). Oxygen support with NRBM resulted in a reduced all mortality rate over HFNO (26.2% versus 45%) but the mortality rate after treatment failure in either group (HFNO or NRBM) remained high (91% versus 92%). Conclusion: Oxygen support with NRBM results in both reduced device failure rate and higher survival among patients of COVID-19 with AHRF.

Molecular Characterization and Identification of Potential Inhibitors for 'E' Protein of Dengue Virus.

Dengue is an arthropod-borne acute febrile illness caused by Dengue Virus (DENV), a member of Flaviviridae. Severity of the infection ranges from mild self-limiting illness to severe life-threatening hemorrhagic fever (DHF) and dengue shock syndrome (DSS). To date, there is no specific antiviral therapy established to treat the infection. The current study reports the epidemiology of DENV infections and potential inhibitors of DENV 'E' protein. Among the various serotypes, DENV-2 serotype was observed more frequently, followed by DENV-4, DENV-1, and DENV-3. New variants of existing genotypes were observed in DENV-1, 2, and 4 serotypes. Predominantly, the severe form of dengue was attributable to DENV-2 infections, and the incidence was more common in males and pediatric populations. Both the incidence and the disease severity were more common among the residents of non-urban environments. Due to the predominantly self-limiting nature of primary dengue infection and folk medicine practices of non-urban populations, we observed a greater number of secondary dengue cases than primary dengue cases. Hemorrhagic manifestations were more in secondary dengue in particularly in the pediatric group. Through different computational methods, ligands RGBLD1, RGBLD2, RGBLD3, and RGBLD4 are proposed as potential inhibitors in silico against DENV-1, -2, -3, and -4 serotypes.

Effect of delay in processing and storage temperature on diagnosis of SARS-CoV-2 by RTPCR testing.

PURPOSE: A large number of new molecular or virology laboratories have been established to increase the testing capacity for SARS-CoV-2. Due to heavy workload, there is delay in testing of samples. In order to avoid the negative effect of delayed testing on RTPCR results guidelines are issued from WHO and CDC to transport samples in cold chain. However, in pandemic situations the recommended guidelines for transport and storage conditions are often compromised. This study was conducted to evaluate the effect of sample storage conditions at different temperatures on the results of RT PCR test. METHODS: Total 275 samples were included in this study, among these 126 samples tested positive and 149 samples tested negative. All samples were aliquoted into two and the aliquotes stored in duplicate at 4 â€‹°C and room temperature. All aliquots stored at both the temperatures were tested by RTPCR every 24 hours up to 5 days. RESULTS: Diagnostic accuracy decreased from day1 to day 5 at both the storage temperatures i,e 4 â€‹°C and room temperature in comparison to the initial day results. Positivity decreased on an average of 9.02% at 4 â€‹°C and at 9.27% at room temperature per day. Among total 126 positive samples on an average false negative and failure of internal control at 4 â€‹°C and room temperature was 8.86%, 8.22% and 3.64%, and 4.12%, respectively. All the samples with CT value â€‹< â€‹30 remained positive at both temperatures up to 5 days. Few samples with >30 CT value showed variable results i.e. positive, negative or internal control failure from day 1 (2nd day after sample collection) onwards. CONCLUSIONS: There was no significant difference between RT PCR results of samples stored at 4 â€‹°C and room temperature up to 5 days of collection. However internal control failure was more in samples stored at room temperature. Therefore, samples received without cold chain also may be processed by RTPCR and should not be rejected.

E6-E7 based nested multiplex PCR assay for genital HPV detection and simultaneous typing of 15 high and low-risk HPV types.

PURPOSE: Due to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping. METHODS: HPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 1010genome equivalents (GE). DNA sequencing was done to confirm the PCR results. RESULTS: The assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results. CONCLUSIONS: The designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.

An Epidemiological Analysis of SARS-CoV-2 Genomic Sequences from Different Regions of India.

The number of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cases is increasing in India. This study looks upon the geographic distribution of the virus clades and variants circulating in different parts of India between January and August 2020. The NPS/OPS from representative positive cases from different states and union territories in India were collected every month through the VRDLs in the country and analyzed using next-generation sequencing. Epidemiological analysis of the 689 SARS-CoV-2 clinical samples revealed GH and GR to be the predominant clades circulating in different states in India. The northern part of India largely reported the 'GH' clade, whereas the southern part reported the 'GR', with a few exceptions. These sequences also revealed the presence of single independent mutations-E484Q and N440K-from Maharashtra (first observed in March 2020) and Southern Indian States (first observed in May 2020), respectively. Furthermore, this study indicates that the SARS-CoV-2 variant (VOC, VUI, variant of high consequence and double mutant) was not observed during the early phase of virus transmission (January-August). This increased number of variations observed within a short timeframe across the globe suggests virus evolution, which can be a step towards enhanced host adaptation.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

Evaluation of loop-mediated isothermal amplification assay for detection and typing of human papilloma virus 16 and 18 from endocervical samples.

Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.

Parathyridaria percutanea and Subcutaneous Phaeohyphomycosis.

Parathyridaria percutanea is an emerging fungus causing subcutaneous phaeohyphomycoses in renal transplant recipients in India. We identified P. percutanea from a patient with subcutaneous phaeohyphomycosis. From our culture collection, we identified the same fungus from 4 similar patients. We found 5 cases previously described in literature.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India. Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases. Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups. Interpretation & conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Co-circulation of four dengue serotypes at South Eastern Andhra Pradesh, India: A prospective study.

Background: Dengue is one of the most important mosquito-borne viral diseases in the world. The emergence and spread of four dengue viruses (DENVs) (serotypes) represent a global pandemic. The four distinct serotypes are, namely, DENV-1, DENV-2, DENV-3 and DENV-4. Very few dengue serotyping studies have been reported from Andhra Pradesh. In this context, the present study focuses on the circulating serotypes of dengue in South-Eastern Andhra Pradesh. Methodology: Study was done at Sri Venkateswara Institute of Medical Sciences, a teaching hospital in Tirupati, Andhra Pradesh. Acute phase dengue serum samples were collected and tested for NS1 antigen and anti-human IgM antibodies by enzyme-linked immunosorbent assay (ELISA). NS1-positive samples were further serotyped by reverse transcriptase real-time polymerase chain reaction (rRT-PCR). Results: A total of 398 serum samples were received from clinically suspected dengue fever cases. Of these, 150 (37.7%) samples were positive for NS1 and/or IgM ELISA. The 96 NS1 antigen-positive samples were further processed for serotyping, of which 36 were negative by rRT-PCR. DENV-2 (41%) was the predominant serotype, followed by DENV-4 (37%), DENV-3 (12%) and DENV-1 (10%) in descending order. Conclusion: This study reports the all four dengue serotypes' co-circulation. This is the first report from South Eastern Andhra Pradesh. Amongst four, DENV-2 was predominant followed by DENV-4. The information of predominant serotypes can guide in forecasting dengue outbreaks and improving control measures of vectors thus may be helpful in the prevention of outbreaks.

Subcutaneous Rhytidhysteron Infection: A Case Report from South India with Literature Review.

Rhytidhysteron is a saprophytic dematiaceous fungus which rarely infects humans. Though virtually all individuals are exposed, very few develop the disease. Only seven human cases are reported till date. The present case is the second case from South India. A 40-year-old immunocompetent female agricultural worker, presented with a swelling on the dorsum of the right hand. Fine needle aspiration cytology (FNAC) of the swelling revealed short, thick, branched septate fungal hyphae. The isolate was moderately slow growing; grayish white colonies were observed on Sabouraud's Dextrose Agar (SDA) slant. On further incubation, the colonies turned floccose, greyish black and the black pigment was observed on the reverse. Microscopy of lactophenol cotton blue tease mount showed thick, brown septate hyphae without any fruiting bodies. Molecular typing confirmed the isolates as Rhytidhysteron rufulum. Identification of all clinical isolates of nonsporulating fungi to genus level is necessary to identify rare fungi infecting humans.

A study of influenza 2017-2018 outbreak in Andhra Pradesh, India.

BACKGROUND AND OBJECTIVES: Influenza virus is a typical human pathogen causing serious respiratory illness resulting in significant mortality throughout the globe. Andhra Pradesh witnessed the first case of influenza A H1N1 in India from Hyderabad (now in Telangana) on May 16, 2009. In the recent past, Andhra Pradesh witnessed exponential increase in the number of confirmed cases of influenza infection. In this study, we present the salient features of the recent outbreak of influenza during 2017-2018 in the state of Andhra Pradesh, first of its kind after the division of the state. MATERIALS AND METHODS: Clinically, suspected cases of influenza-like illness received in the Virus Research and Diagnostic Laboratory, Department of Microbiology, Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupati, from January 2017 to May 2018 were included in the study. The samples were tested for influenza A, influenza A (H1N1) pdm09, influenza A (H3N2), influenza B, influenza B/Yamagata and influenza B/Victoria. RESULTS: A total of 1286 samples were received for testing. The positive samples were influenza A unsubtypable (109), influenza A (H1N1) pdm09 (356), influenza A (H3N2) (38) and influenza B (19; Victoria - 2, Yamagata - 17). There was no significant difference in positivity between genders with 260 (49.81%) females and 262 (50.19%) males being positive. CONCLUSION: The outbreak started in the late monsoon (January) of 2017 and had two peaks; one in summer months and another in winter months. Influenza B virus was reported from December 2017 to May 2018. Age groups ≤5 years and 6-18 years had higher positivity as compared to other age groups. Regular surveillance programmes are required for assessing the trends of influenza infections due to various subtypes and to plan timely and adequate steps for preventing the spread to larger vulnerable population.

Antibiotic resistance & pathogen profile in ventilator-associated pneumonia in a tertiary care hospital in India.

BACKGROUND & OBJECTIVES: Ventilator-associated pneumonia (VAP) is an important hospital-acquired infection with substantial mortality. Only a few studies are available from India addressing the microbiological aspects of VAP, which have been done with small study populations. This study was carried out in the intensive care units (ICUs) of a tertiary care hospital to assess the profile of pathogens and to determine the pattern of antimicrobial resistance. METHODS: This was a retrospective study of clinically suspected cases of VAP. Over a three year period, a total of 247 cases in 2011, 297 in 2012 and 303 in 2013 admitted in ICUs on mechanical ventilation with clinical evidence of VAP were included in our study. The endotracheal aspirate samples from these suspected cases were subjected to quantitative culture technique, and colony count of ≥10[5] colony forming units/ml was considered significant. Antimicrobial susceptibility test for the isolates was done. RESULTS: VAP rates of 44.1, 43.8 and 26.3 were seen in 2011, 2012 and 2013, respectively. In all the three years, non-fermentative Gram-negative bacilli were the predominant organisms, followed by Pseudomonas spp. and Klebsiella spp. Staphylococcus aureus exhibited a downwards trend in prevalence from 50.0 per cent in 2011 to 34.9 per cent in 2013. An increase in vancomycin-resistant enterococci was seen from 4.3 per cent in 2012 to 8.3 per cent in 2013, while methicillin resistance amongst the S. aureus crossed the 50 per cent mark in 2013. An increasing trend in resistance was shown by Pseudomonas spp. for piperacillin-tazobactam (PTZ), amikacin and imipenem (IPM). For the non-fermenters, resistance frequency remained very high except for IPM (33.1%) and polymyxin-B (2.4%). INTERPRETATION & CONCLUSIONS: Our findings show VAP as an important problem in the ICU setting. The incidence of multidrug-resistant pathogens was on the rise. The resistance pattern of these pathogens can help an institution to formulate effective antimicrobial policy. To have a comprehensive pan-India picture, multicentric studies are needed.

Molecular characterization of Orientia tsutsugamushi serotypes causing scrub typhus outbreak in southern region of Andhra Pradesh, India.

BACKGROUND & OBJECTIVES: Scrub typhus is a vector-borne zoonotic infection caused by Orientiatsutsugamushi. Local epidemiology of the circulating serotypes of scrub typhus is not available from most parts of India. We conducted this study for the diagnosis of scrub typhus using IgM ELISA and to detect O. tsutsugamushi serotypes circulating in southern Andhra Pradesh, India. METHODS: Samples were collected from patients clinically suspected to have scrub typhus and were subjected to IgM ELISA to measure IgM antibodies against O. tsutsugamushi. Nested polymerase chain reaction (PCR) was performed targeting strain-specific regions in ELISA-positive samples. RESULTS: Of a total of 663 samples, 258 (38.91%) were found to be positive by IgM ELISA. Serotypes could be detected in 230 (34.69%) samples only. Only two serotypes, Karp and Kawasaki, were found in the serum samples, with the former being predominant. The dual infection of Karp and Kawasaki serotypes was found in seven patients. Other serotypes such as Gilliam, Kuroki and Kato were not detected in the samples. INTERPRETATION & CONCLUSION: The nested PCR products proved useful in presumptively identifying the endemic O. tsutsugamushi serotypes. The present study could be significant in understanding scrub typhus epidemiology in this region.