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Objectives: Pneumocystis pneumonia (PCP) remains a debilitating cause of death among HIV-infected patients. The combination trimethoprim/sulfamethoxazole (SXT) is the most effective anti-Pneumocystis treatment and prophylaxis. However, long-term use of this combination has raised alarms about the emergence of resistant organisms. This study was performed to investigate mutations in the dihydropteroate synthase (DHPS) gene and their clinical consequences in HIV-infected patients with PCP., Methods: A total of 76 clinically suspected cases of PCP among HIV-seropositive adult patients from March 2014 to March 2017 were included. Clinical samples (bronchoalveolar lavage fluid and sputum) were investigated for the detection of Pneumocystis jirovecii using both microscopy and nested PCR. DHPS genotyping and mutational analyses were performed and the data were correlated with clinical characteristics., Results: Among the 76 enrolled HIV-positive patients, only 17 (22.4%) were positive for P. jirovecii. DHPS gene sequencing showed a novel nucleotide substitution at position 288 (Val96Ile) in three patients (3/12; 25.0%). Patients infected with the mutant P. jirovecii genotype had severe episodes of PCP, did not respond to SXT and had a fatal outcome (P=0.005). All three patients had a CD4 + T-cell count <100 cells/μL, and two also had co-infections., Conclusion: This study suggests that the emergence of a mutant P. jirovecii genotype is probably associated with drug resistance and mortality. The data also suggest that DHPS mutational analyses should be performed in HIV-seropositive patients to avoid treatment failure and death due to PCP. However, the role of underlying disease severity and co-morbidities should not be underestimated., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Data on the genetic diversity of Pneumocystis jirovecii causing Pneumocystis pneumonia (PCP) among children are still limited, and there are no available data from the Indian subcontinent, particularly associations between genotypes and clinical characteristics. A total of 37 children (62 days-12 years [median 5.5 years]) were included in this study. Pneumocystis was diagnosed by microscopy using Grocott-Gomori methenamine silver stain in 12 cases and by nested PCR using mtLSUrRNA in 25 cases. Genotyping was performed using three different genes, mitochondrial large subunit ribosomal RNA (mtLSUrRNA), dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR). mtLSUrRNA genotype 3 and novel mutations at the gene target DHFR (401 T > C) and DHPS 96/98 were frequently observed and clinically associated with severe PCP and treatment failure. Phylogenetic analyses revealed 13 unique sequence types (STs). Two STs (i) 3-DHFR 401 T > C-DHPS 96/98 - PJ1 and (ii) 3-DHFR 401 T > C-DHPS 96- PJ3 were significantly associated with treatment failure and high mortality among PCP-positive patients. In conclusion, the present study strongly suggests the emergence of virulent P. jirovecii strains or genetic polymorphisms, leading to treatment failure and high mortality. Our study is the first of its kind from the Indian subcontinent and has highlighted the genetic diversity of Pneumocystis jirovecii among children and their clinical outcomes. These findings emphasize the need to focus more on genotypes to better understand the epidemiology of Pneumocystis pneumonia.

The present study was carried out with the objectives of genotyping Pneumocystis jirovecii at three distinct loci, to identify the single nucleotide polymorphisms (SNPs), and to study its clinical implications in patients with Pneumocystis pneumonia (PCP). Analysis of genetic diversity in P. jirovecii from immunocompromised patients was carried out by genotyping at three distinct loci encoding mitochondrial large subunit rRNA (mtLSU rRNA), cytochrome b (CYB), and superoxide dismutase (SOD) using polymerase chain reaction (PCR) assays followed by direct DNA sequencing. Of the 300 patients enrolled in the present study, 31 (10.33%) were positive for PCP by a specific mtLSU rRNA nested PCR assay, whereas only 15 P. jirovecii could be amplified at the other two loci (SOD and CYB). These positives were further subjected to sequence typing. Important genotypic combinations between four SNPs (mt85, SOD110, SOD215, and CYB838) and clinical outcomes could be observed in the present study, and mt85A, mt85T, and SOD110C/SOD215T were frequently associated with "negative follow-up". These SNPs were also noted to be relatively more prevalent amongst circulating genotypes in our study population. The present study is the first of its kind from the Indian subcontinent and demonstrated that potential SNPs of P. jirovecii may possibly be attributed to the clinical outcome of PCP episodes in terms of severity or fatality in different susceptible populations likely to develop PCP during their course of illness.

Introduction: Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India., Methodology: Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms., Results: Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and 10% (18/180) were positive by mt LSU rRNA PCR assay. Of these 18 positive samples, only 77% (14/18) were amplified by the DHFR gene PCR assay. A total of 16 nucleotide substitutions were observed in 42% (6/14) samples targeted for the DHFR gene, of which 8 nucleotide substitutions were synonymous and the rest were non-synonymous., Conclusions: The DHFR gene mutations found in this study may possibly indicate an association of process likely to contribute to therapeutic failure or an evolutionary process, and warrant continuous monitoring.

Pneumocystis jirovecii is an opportunistic pathogen that causes severe pneumonia in immunocompromised patients. To study the genetic diversity of P. jirovecii in India the upstream conserved sequence (UCS) region of Pneumocystis genome was amplified, sequenced and genotyped from a set of respiratory specimens obtained from 50 patients with a positive result for nested mitochondrial large subunit ribosomal RNA (mtLSU rRNA) PCR during the years 2005-2008. Of these 50 cases, 45 showed a positive PCR for UCS region. Variations in the tandem repeats in UCS region were characterized by sequencing all the positive cases. Of the 45 cases, one case showed five repeats, 11 cases showed four repeats, 29 cases showed three repeats and four cases showed two repeats. By running amplified DNA from all these cases on a high-resolution gel, mixed infection was observed in 12 cases (26.7%, 12/45). Forty three of 45 cases included in this study had previously been typed at mtLSU rRNA and internal transcribed spacer (ITS) region by our group. In the present study, the genotypes at those two regions were combined with UCS repeat patterns to construct allelic profiles of 43 cases. A total of 36 allelic profiles were observed in 43 isolates indicating high genetic variability. A statistically significant association was observed between mtLSU rRNA genotype 1, ITS type Ea and UCS repeat pattern 4., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Opportunistic infections (OIs) are a leading cause of mortality and morbidity in patients living with HIV/AIDS. Data on the proper administration of prophylactic regimes for the prevention of OIs in such patients are scarce. A total of 205 confirmed HIV-infected patients were enrolled in the study from the inpatient wards and outpatient services. The treatment given to them for the prevention of Pneumocystis carinii (jiroveci) pneumonia was compared with the established guidelines and the proportions of those receiving proper treatment were calculated. Primary prophylaxis was seen to be satisfactory in the case of P. carinii (jiroveci) pneumonia. The prophylaxis was not given properly for tuberculosis and other common OIs. Secondary prophylaxis was up to the mark. Prophylaxis in AIDS patients seems to be a major problem area and a lot of efforts need to be directed toward it since patients suffering from AIDS are bound to have a downhill course despite provision of all available treatment options.

Pneumocystis pneumonia (PCP), a common and serious opportunistic infection in immunocompromised patients, is caused by Pneumocystis jirovecii (formerly known as Pneumocystis carinii f. sp. hominis). The aim of the present study was to describe the prevalence and distribution of genotypes of P. jirovecii based on sequence polymorphisms at mitochondrial large subunit ribosomal RNA (mt LSU rRNA) region in both HIV and non-HIV immunocompromised individuals with a positive PCR result for PCP in a tertiary health care centre in northern India. From January 2005 to October 2008, 50 patients [22 HIV-seropositive individuals, 10 post-renal transplant (PRT) recipients, 3 cancer patients, and 15 patients with various other kinds of immunosuppression] were found to be positive for P. jirovecii using PCR at the mt LSU rRNA gene. Genotyping of the positive samples was performed at the mt LSU rRNA locus. Genotype 2 was the most common accounting for 42% of total types. This was followed by the genotypes 3 (24%), 1 (20%), and 4 (8%). Mixed infection was observed in 3 cases (6%). The rates of genotype distribution were similar in HIV-seropositive individuals, cancer patients, and in patients with other kinds of immunosuppression. In the PRT recipients, genotype 1 was the most prevalent type (80%). This is the first study describing the prevalence of genotypes in HIV-infected and HIV-uninfected, immunocompromised patients based on the mt LSU rRNA gene from the Indian subcontinent. The most prevalent genotype observed was type 2 in contrast to many studies from other parts of the world where genotype 1 was the most prevalent type, suggesting geographical variation., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Studies on Pneumocystis jirovecii dihydropteroate synthase (DHPS) genotypes among non-HIV immunocompromised patients from developing countries are rare. In the present prospective investigation, 24 (11.8%) cases were found to be positive for Pneumocystis jirovecii out of 203 non-HIV patients with a clinical suspicion of Pneumocystis pneumonia (PCP). Dihydropteroate synthase (DHPS) genotype 1 (Thr55+Pro57) was noted in 95.8% P. jirovecii isolates in the present study in contrast to only 4.1% of patients with DHPS genotype 4 (Thr55Ala + Pro57Ser).

Introduction: Pneumocystis jirovecii dihydropteroate synthase (DHPS) gene mutations' (55th and 57th codon) association with prior sulfa prophylaxis failure has been reported from both developed and developing countries. We conducted a prospective study to determine the prevalence of P. jirovecii DHPS mutations from 2006 to 2009 on P. jirovecii isolates obtained from HIV-infected patients with a clinical diagnosis of Pneumocystis carinii pneumonia (PCP) admitted to our tertiary care reference health center in New Delhi, India., Methodology: Detection of P. jirovecii cysts was performed by direct fluorescent antibody (DFA) staining and by Grocott's-Gomori methenamine silver staining (GMS). DNA detection was performed by polymerase chain reaction (PCR) using primers for the major surface glycoprotein (MSG) gene. P. jirovecii DHPS gene was amplified by nested PCR protocol and sequenced for detecting mutations at the 55th and 57th codons., Results: Out of 147 HIV-positive patients with suspected Pneumocystis pneumonia (PCP), 16 (10.8%) PCP positive cases were detected. Of 16 cases, nine (56.2%) were positive by DFA staining, four (25%) were positive by Grocott's-Gomori methenamine silver staining, and all 16 were positive by MSG PCR. DHPS mutations at the 55th and 57th codons were observed in 6.2% of HIV patients studied, which was relatively low compared to reports from developed nations., Conclusions:   Prevalence of Pneumocystis jirovecii DHPS mutations associated with cotrimoxazole treatment failure may be low in the Indian subpopulation of HIV-positive patients and warrants larger studies to elucidate the true picture of Pneumocystis jirovecii sulfa drug resistance in India.

Pneumocystis jirovecii is the cause of Pneumocystis pneumonia (PCP) in immuno-compromised individuals. The aim of this study was to describe the genotypes/haplotypes of P. jirovecii in immuno-compromised individuals with positive polymerase chain reaction (PCR) result for PCP. The typing was based on sequence polymorphism at internal transcribed spacer (ITS) regions of rRNA operon. Phylogenetic relationship between Indian and global haplotypes was also studied. Between January 2005 to October 2008, 43 patients were found to be positive for Pneumocystis using PCR targeting mitochondrial large subunit rRNA (mt LSU rRNA) and ITS region. Genotyping of all the positive samples was performed at the ITS locus by direct sequencing. Nine ITS1 alleles (all previously known) and 11 ITS2 alleles (nine previously defined and two new) were observed. A total of 19 ITS haplotypes, including five novel haplotypes (DEL1r, Edel2, Hr, Adel3 and SYD1a), were observed. The most prevalent type was SYD1g (16.3%), followed by types Ea (11.6%), Ec (9.3%), Eg (6.9%), DEL1r (6.9%), Ne (6.9%) and Ai (6.9%). To detect mixed infection, 30% of the positive isolates were cloned and 4-5 clones were sequenced from each specimen. Cloning and sequencing identified two more haplotypes in addition to the 19 types. Mixed infection was identified in 3 of the 13 cloned samples (23.1%). Upon construction of a haplotype network of 21 haplotypes, type Eg was identified as the most probable ancestral type. The present study is the first study that describes the haplotypes of P. jirovecii based on the ITS gene from India. The study suggests a high diversity of P. jirovecii haplotypes in the population., (Copyright 2010 Elsevier B.V. All rights reserved.)

Objective: To study the role of cytology and polymerase chain reaction (PCR) for the detection of Pneumocystis carinii pneumonia (PCP) in bronchoalveolar lavage fluid (BALF)., Study Design: In a prospective observational study, a BALF pellet from 55 patients (35 immunosuppressed patients clinically suspicious for PCP and 20 immunocompetent individuals not suspicious for PCP) were subjected to cytology examination as well as PCR for PCP using the major surface glycoprotein gene. Additionally, DNA was extracted from destained cytology slide scrapings from 5 cases each positive for PCR and negative for PCP on BALF, respectively, and was further subjected to PCR., Results: Of 35 immunosuppressed patients, 2 were positive on microscopy for PCP, while 5 (including 2 microscopy positive) were positive by PCR from BALF. Four of the 5 positive cases showed predominantly alveolar macrophages, while one showed 40% alveolar macrophages, 30% lymphocytes and 20% neutrophils upon cellular analysis of BALF. One case had coinfection with Cryptococcus. Destained smear scrapings of PCR-positive slides (n = 5) were all successfully amplified for PCP major surface glycoprotein PCR after DNA extraction, while control slides (n = 5) did not show amplification., Conclusion: BALF is considered the sample of choice for the diagnosis of PCP. In our study, cases with PCP showed mainly alveolar macrophages on BALF examination by cytology and no or mild inflammatory cells. Thus, inflammatory cell population found on cytology may provide direction for PCR. Two-step screening of BALF using cytology followed by PCR from slides is possible and may overcome the limitations of BALF cytology alone for the diagnosis of PCP.

A total of 327 clinical specimens, including both invasive and noninvasive samples, obtained from 275 patients with various types of underlying immunocompromised conditions and a clinical suspicion of Pneumocystis pneumonia (PCP) were subjected to 2 different nested polymerase chain reaction (PCR) assays. The target genes used for nested PCR were mitochondrial large subunit ribosomal RNA (mtLSU rRNA) and internal transcribed spacer (ITS) region. The results were compared with a single-round PCR targeting major surface glycoprotein (MSG) gene. Amplification was successful in 16% of cases by mtLSU rRNA nested PCR, in 14.5% by ITS nested PCR, and in 10.9% by MSG PCR. The nested mtLSU rRNA PCR was found to be more sensitive (100% sensitive and 98.7% specific) and useful in detecting PCP for its use in routine diagnosis in our settings. Thus, this assay may be quite useful in the identification of patients who are in the early stage of Pneumocystis jirovecii infection with an organism load that could not be easily detected by the single-step PCR.

Background & Objectives: Pneumocystis jiroveci (also known as P. carinii) causes fatal pneumonia in patients with AIDS and other immunocompromised patients. Co-trimoxazole (trimethoprim + sulphamethoxazole, TMP-SMZ) is the drug of choice for treatment and prophylaxis. Widespread use of sulpha medication has raised the possible selection of resistant P. jiroveci strains worldwide. Non-synonymous polymorphisms associated with sulpha resistance have been observed in P. jiroveci dihydropteroate synthase (DHPS) gene at codons 55 and 57. In view of this, we investigated mutation at DHPS locus amongst P. jiroveci isolates obtained at a tertiary care hospital in north India., Methods: Microscopic examination of P. jiroveci in 69 clinical samples obtained from patients suspected to have P. carinii pneumonia (PCP), was performed by Grocott's Gomori methenamine silver and direct fluorescent antibody staining. Molecular studies were carried out by polymerase chain reaction (PCR) using major surface glycoprotein (MSG) as the target gene. Investigations for DHPS mutations were carried at specific 55th and 57th codon using PCR-RFLP (restriction fragment length polymorphism) assay., Results: Microscopic examination detected P. jiroveci in four cases and MSG gene was amplified in five cases. Further, amplification of DHPS gene was successful in four of the five cases positive by MSG gene PCR. No point mutation was observed and all four isolates presented wild-type sequences at DHPS gene by RFLP analysis., Interpretation & Conclusion: Although our findings suggest that in Indian subpopulation, point mutations in DHPS gene of P. jiroveci are not as common as in other parts of the developed world, further studies are needed.

Background: Pneumocystis carinii pneumonia (PCP), caused by opportunistic agent Pneumocystis jirovecii (formerly, Pneumocystis carinii is one of the most serious respiratory infection in immunocompromised patients., Aim: The present study was conducted to compare polymerase chain reaction (PCR) assays targetting three different genes of Pneumocystis to study their application in its diagnosis., Methods: One hundred and eighty (n = 180) clinical samples from 145 immunocompromised patients with clinical suspicion of PCP and 35 samples from control group of 30 immunocompetent individuals with respiratory infections other than PCP were prospectively examined for the presence of Pneumocystis jirovecii (P. jirovecii). All the samples were subjected to microscopic examination, one single [major surface glycoprotein, (MSG)] and two nested [mitochondrial large subunit ribosomal ribonucelic acid, (mtLSU rRNA) and internal transcribed spacer (ITS) region], polymerase chain reaction assays., Results: Microscopic examination was positive in only six (n = 6) patients, whereas single round MSG PCR detected P. jirovecii deoxyribonucleic acid (DNA) in 16 cases. When the clinical samples were tested by mtLSU rRNA and ITS nested PCR assays, it was possible to detect seven additional cases of PCP, making it to a total of 23 cases. None of the clinical specimens in control group (n = 30) were positive by any of the above-mentioned techniques. Amongst the 81 bronchoalveolar lavage (BAL) samples tested, 16 were positive by MSG PCR, while 20 were positive by both nested, i.e., mtLSU rRNA and ITS PCR assays. Similarly, out of 50 sputum samples, only three were positive by MSG, seven by mtLSU rRNA and six by ITS nested PCR assays., Conclusion: It has been observed that MSG is relatively more sensitive when single round PCR assay is used for detection of human Pneumocystosis compared to the first (single) rounds of either ITS or mtLSU rRNA nested PCRs. However, the two nested PCRs using ITS and mtLSU rRNA have been found to be more sensitive. On comparison of two nested PCR assays, the results have been more or less comparable.

We prospectively examined 143 clinical samples from 115 patients including both HIV infected (n=53) and HIV uninfected immunocompromized (n=62) patients, with lung infiltrates and with clinical features suggestive of Pneumocystis carinii pneumonia/ PneumoCystis Pneumonia (PcP), using both microscopic techniques as well as PCR assay. Clinical samples in the present study consisted of bronchoalveolar lavage (BAL), tracheal aspirate (TA), nasopharyngeal aspirate (NPA), sputum and gastric aspirate (GA). Another group of 21 individuals with other respiratory diseases not compatible with PcP served as control during the study period of 15 months. Overall, P. jirovecii positivity rate by PCR was 12.17% (14/115 patients) compared to 3.4% (4/115) by microscopy. None of the specimens in the control group was positive by any of the techniques used. All PCR negative patients including cases and controls showed no evidence of PcP. After resolution of the discrepant results upon review of the clinical data, the sensitivity and specificity were 100% and 99%, respectively, for PCR and 30.7% and 100%, respectively, for microscopy by GMS staining. Thus, our data support the significance of PCR assay for confirming and improving the diagnosis of PcP in high-risk patients.

Pneumocystis is an atypical fungus causing pneumonia in immuno-compromised individuals. Though previously termed as Pneumocystis carinii, the recent taxonomy has considered human derived Pneumocystis to be a different species Pneumocystis jiroveci. The organism is the most common cause of opportunistic infections among patients with acquired immunodeficiency syndrome (AIDS) in developed countries. Incidence of Pneumocystis pneumonia or pneumocystosis in developing countries including India continues to be low. Microscopy of appropriate clinical samples has been the mainstay of diagnosis of pneumocystosis. Amplification techniques are now being evaluated for detection of P. jiroveci. This review attempts to give a recent update on P. jiroveci with special focus on epidemiology, taxonomy, current diagnostic modalities and recommended immuno-prophylaxis.