Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania and sand flies co-exist.

Visceral leishmaniasis caused by Leishmania infantum in Thailand.

Thailand is considered as a non-endemic area for leishmaniasis. We report the first case of visceral leishmaniasis caused by Leishmania infantum in a Thai man living in Bangkok.

Genetic diversity of Pneumocystis jirovecii in Thailand.

A fungus Pneumocystis jirovecii, which causes a diffuse bilateral pneumonia called Pneumocystis pneumonia (PcP) is one of the most common opportunistic infections in HIV-infected patients in Thailand. Molecular techniques have demonstrated diversity among isolates of P. jirovecii by comparison of DNA-sequence variation at the internal transcribed spacer region 1 (ITS1) and region 2 (ITS2) of the nuclear ribosomal RNA genes. The studies confirm that a high diversity of P. jirovecii ITS types exists in different populations from different geographical areas. Type Eg is found globally from represent countries in Europe, North America, South Africa and Asia. Among the 23 types of P. jirovecii observed in Thailand, type Ir is present at the highest frequency (28.6 %), followed by type Eb (21.4%) and types Eg and Rp (14.3 %), respectively. Ir and Rp are unique types observed in Thailand. Mixed infections of more than one types of P. jirovecii are commonly observed in all studies with prevalence of 25-82 %. Moreover, unique types of P. jirovecii can be found in a specific group of populations. These types may be used as genetic markers to study the evolution of the organism in each geographical area.

Pneumocystis jiroveci in HIV/AIDS patients: detection by FTA filter paper together with PCR in noninvasive induced sputum specimens.

OBJECTIVES: To detect P. jiroveci (previously named P. carinii) by PCR using FTA filter paper to extract the DNA, from noninvasive induced sputum samples of HIV/AIDS patients. MATERIAL AND METHOD: Fifty two HIV/AIDS patients suspected of Pneumocystis jiroveci pneumonia (PJP) in King Chulalongkorn Memorial Hospital were recruited. Both cytological method and PCR with FTA filter paper technique were performed to detect P jiroveci from each specimen. RESULTS: The detectability rate of P. jiroveci infection was 21%. The PCR with FTA filter paper method was 4 folds much more sensitive than Giemsa staining technique. P. jiroveci was detected in 18% of the HIV/AIDS patients in spite of receiving standard PJP prophylaxis. CONCLUSION: Detection of P. jiroveci by using FTA filter paper together with PCR in induced sputum samples could detect more cases of P. jiroveci infection than by using cytological method. DNA extraction using the FTA filter paper was more rapid and convenient than other extraction methods. The causes of failure of PJP prophylaxis should be evaluated.